CA2003408A1 - Antiviral agents - Google Patents

Abstract

ABSTRACT OF THE DISCLOSURE

Nucleoside compounds of the formula wherein:
B is guanine, adenine, thymine, uracil, cytosine, hypoxanthine, xanthine, 5-methylcytosine, 4-ethoxy-5-methyl-2-oxo-pyrimidine, 4-isopropoxy-5-methyl-2-oxo-pyrimidine, or 5-methyl-2-oxo-pyrimidine;
X is H or F;
Y' is H, OH, OCH3 or F; and Z is or

Description

Z(~ 408 ANTIVIRAI. AGENTS

Field of the Invention The present invention relates to antiviral agents, particularly to nucleoside-based antiviral drugs, and specifically to a series of 2'-deosy-4'-azido-substituted nucleosides. The invention is also directed to ormulations and methods for treating viral infections in a mammal, as well as to methods of making the subject compounds.

Ba~kg~ound Information Viruses have long been known to be the cause of some of the most costly, troublesome and devastating infections to man. In recent years, this pattern has been underscored by the onset of Acquired Immune Deficiency Syndrome (AIDS), which has been found to be the result of infection by the human immunodeficiency virus (HIV).
Various active agents have been proposed for the treatment of viruses such as AIDS. Typicall~, these active agents have suffered from a disadvantageous therapeutic indes, i.e., the ratio of activity to toxicity (in other words, their beneficial effect was outweighted by their tosic nature).

0733M (now 07330) 26530-FF

, .

2C~)~408 For esample, the drug AZT (3'-azidothymidine) is described in European Patent Application 86307071.0; it is presently used for treatment of AIDS. It is not, however, a cure for the disease. AZT is also fairly toxic to the bone marrow, requiring patients under treatment to receive frequent blood transfusions, and although their disease symptoms are diminished and life is prolonged, AIDS related death is still considered inevitable.
Another e~ample is the drug DDC ~2',3'-dideoxy-cytidine), as described in PCT/US86/01626, having an international filing date of August 8, 1986, claiming priority from U.S. Serial No. 769,017, filed August 26, 1985. This drug is currently under investigation for the treatment of HIV infection. It is more potent than AZT, but, it is also very toxic, leading often to severe peripheral neuropathy.
4'-Substituted nucleosides have been described previously [see Ann. N.Y. Acad. Sci., ~, 151 (1975)].
More particularly, various 4'-methosypurine and 4'-methosypyrimidine ribonucleosides and 4'-azidocytidine have been synthesized and screened for their antiviral activity, but, have not shown any usefulness in this regard. For esample, 4'-azidocytidine is cytotoxic and devoid of anti-HIV activity.
It has remained desired to provide antiviral active agents having a high therapeutic index; that is, provide beneficial effect with little or no toxic effect. The compounds of the present invention provide such beneficial effect with low toxic effect.

SUMMARY OF THE INVENTION

One aspect of the present invention relates to 0733M (now 07330~ 26530-FF

20~3408 2'-deoxy-4'-azido-nucleosides, i.e., the compounds of Formula I:

~ J Formula I
N3¦

Y' wherein:
B is guanine, adenine, thymine, uraci1, cytosine, hypoxanthine, santhine, 5-methylcytosine, 4-etho~y-5-methyl-2-oxo-pyrimidine, 4-isoproposy-5-methyl 2-oso-pyrimidine, or 5-methyl-2-oso-pyrimidine;
X is H or F;
Y' is H, OH, OCH3 or F; and /0 \ O
Z i 8 ~ ~ ~ H2_ or (R ~2 2 OH n where n is zero, one, or three and both R' are hydrogen or lower alkyl; or Y' and Z together form a cyclic phosphate ester;
and the pharmaceutically acceptable esters, ethers and salts thereof.
In another aspect, the invention relates to a pharmaceutical composition comprising a therapeutically e~fective amount of a compound of Formula I or a pharmaceutically acceptable ester or salt thereof admixed with at least one pharmaceutically acceptable excipient.
In still another aspect, the invention relates to a method of treating infections in a mammal by administering to a mammal in need of such treatment a therapeutically effective amount of a compound of 0733M (now 07330) 26530-FF

20~)3408 Formula I or a pharmaceutically acceptable ester or salt thereof.
Yet another aspect of the invention relates to precursors for making the compounds of Formula I and the pharmaceutically acceptable salts and esters thereof, represented by Formula II:
o 7 ~ X~ Formula II

-R
wherein:
B' is the same as 8 in Formula I, or an acylated equivalent thereof;
R is methyl or an acyl group such as anisoyl, benzoyl, acetyl or uran-2-carbonyl;
X i8 H or F; and Z i8 methylene with a leaving group such as iodo or bromo.
Another aspect of the invention relates to processes for making the compounds of Formula I and the pharmaceutically acceptable salts and esters thereof.
For example, a process for preparing a compound of formula I

Z B
L/o~\l Y' wherein:

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20~;~408 8 is guanine, adenine, thymine, uracil, cytosine, hyposanthine, xanthine, 5-methylcytosine, 4-ethosy-5-methyl-2-o~o-pyrimidine, 4-isopropoxy-5-methyl-2-oso-pyrimidine, or 5-methyl-2-o~o-pyrimidine;
X is H or F;
Y' is H, OH, OCH3 or F; and z is N ~ ~ ~2- or ~R'O)2- ~ ~2 -where n is zero, one, or three and both R' are hydrogen or lower alkyl; or Y' and Z together form a cyclic phosphate ester;
and the pharmaceutically acceptable esters, ethers and salts thereof, which comprises a) reacting a compound of formula II

~ ~ Formul3 II
W

O-R
wherein:
B' is the same as B in Formula I, or an acylated equivalent thereof;
R is methyl or an acyl group such as anisoyl, benzoyl, acetyl or furan-2-carbonyl;
X is H or F; and Z is methylene with a leaving group such as iodo or bromo, with an oxidizing agent, followed by the addition of a base; or 0733M (now 07330~ 26530-FF

20~3~0a b) reacting a compound of the formula R -O B
~~ , Y' wherein:
B is guanine, adenine, thymine, uracil, cytosine, hyposanthine, ~anthine, 5-methylcytosine, 4-ethosy-5-methyl-2-oxo-pyrimidine, 4-isopropo~y-5-methy1-2-oxo-pyrimidine, or 5-methyl-2-oxo-pyrimidine;
X is H or F;
Y' is H, OH, OCH3 or F;
R3 is lower alkyl; and R4 i8 -OH or tri-substituted silylo~y;

with (1) if R4 is OH, a silylating agent and a base followed by an azide and a Lewis acid catalyst, or ~2) if R4 is tri-substituted silylosy, reacting an azide and a Lewis acid catalyst; or c) reacting a compound of formula I wherein n is zero, with a phosphorylating agent to give a compound of formula I wherein n is one or three: or d) reacting a compound of formula II with phosphonous acid or a salt of a di-lower alkyl phosphonate to give a compound of formula I wherein Z is (R'O)2P(O)CH2-; or e) reacting a compound of formula I wherein n is one with a cyclizing agent to give a compound of formula I wherein Y' and Z together form a cyclic phosphate; or f) reacting a compound of formula I with a 0733M (now 07330) 26530-FF

.. , . ~

. , ~C~Q8 protecting agent, followed by reaction with a strong base followed by an alkyl halide, followed by deprotection, to give a compound of formula I wherein Y~ is an ether; or g) reacting a compound of formula I with a pyridine catalyst and an acid chloride to give the corresponding ester of a compound of formula I; or h) reacting a compound of formula I with a pharmaceutically acceptable base to form the corresponding base addition salt of formula I; or i) reacting a base addition salt of a compound of formula I with an acid to form the free compound of formula I; or j) converting a pharmaceutically acceptable base addition salt of a compound of formula I to another pharmaceutically acceptable base addition salt of a compound of formula I; or k) convertinq an ester or ether of a compound of formula I to the corresponding free compound of formula I.

~AI~ED DESCRIPTION

Deinitions and General Parameters The followinq definitions are set forth to illustrate and define the meaning and scope of the various terms used to describe the invention herein.
The term ~alkyl~ refers to a cyclic, branched or straight chain monovalent alkyl radical of one to twenty carbon atoms. This term i8 further e~emplified by such radicals as methyl, ethyl, n-propyl, isopropyl, n-butyl, t-butyl, i-butyl, 2-methyl-2-propyl, n-octyl, n-decyl, n-tetradecyl, and n-nonadecyl. The term ~lower alkyl~
refers to a cyclic, branched or straight chain monovalent alkyl radical of one to four carbon atoms. The term "lower alkyl~ is further exemplified by such radicals as 0733M (now 07330) 26530-FF

~ : , 20~3408 methyl, ethyl, n-propyl, isopropyl, n-butyl, t-butyl, or i-butyl.
The term ~lower alkoxy~ refers to the group -O-R ' where R' is lower alkyl.
The term ~acyl~ refers to the group -C(=O)-W wherein W is an alkyl group containing 1 to 20 carbon atoms, adamantyl, aryl, amino, alkylamino, dialkylamino, an alkoxy group containing 1 to 20 carbon atoms, -C~2-O-CH3, -CH2-NH2, or a group of the formula:

~ 2 where A is hydrogen, lower alkyl or aryl.
The term ~aryl~ refers to a monovalent unsaturated aromatic carbocyclic radical having a single ring (e.g., phenyl) or two condensed rings (e.g., naphthyl), which can optionally be mono-, di- or tri-substituted, independently, with hydro~y, lower alkyl, lower alkoxy, chloro, fluoro, and/or cyano.
The term ~halo" refers to fluoro, bromo, chloro and iodo.
The term ~heterocycle~ refers to a monovalent unsaturated or aromatic carbocyclic radical having at least one hetero atom, such as N, O or S, within the ring, each available position of which can optionally be substituted, independently, with, e.g., hydroxy, oxo, amino, imino, lower alkyl, lower alkoxy, bromo, chloro, fluoro, and/or cyano. Included within this class of substituents are purines and pyrimidines.
The term ~purine~ refers to nitrogenous bicyclic heterocycles, typically including the naturally occurring purines adenine (or 6-aminopurine), hypoxanthine (or 6-hydroxypurine), guanine (2-amino-6-oxopurine) and 0733M (now 07330) 26530-FF

." .
, , -- ~ . - - .

200~3~08 xanthine (2,6-dihydroxypurine). These compounds can be of natural or synthetic origin, isolated or manufactured using e~clusively or any combination of chemical, biochemical or enzymological methodology.
The term ~pyrimidine~ refers to nitrogenous monocyclic heterocycles, typically including the naturally occurring pyrimidines cytosine (4-amino-2-oxopyrimidine), uracil (2,4-diosopyrimidine), 5-methylcytosine (4-amino-5-methyl-2-oxopyrimidine), and thymine (5-methyl-2,4-dioxopyrimidine). As used herein, the term pyrimidine also includes moieties that have been derivatized or modified by substitution on the parent skeleton, such as, 4-ethoxy-5-methyl-2-oxo-pyrimidine, 4-isopropo~y-5-methyl-2-oxo-pyrimidine, and 5-methyl-2-oxo-pyrimidine and the like. These compounds can be of natural or synthetic origin, isolated or manufactured using esclusively or any combination of chemical, biochemical or enzymological methodology.
~ Thymidine~ is by deinition 1-(2-deosy-~-D-erythro-pentofuranosyl)thymine. By convention, the compound is not referred to as 2'-deoxythymidine even though the 2 position in Formulae I and II is not OH.
The term ~nucleoside~ refers to a compound composed of any pentose moiety attached to the natural position of a purine (the 9-position) or pyrimidine (the l-position) or to the equivalent position in an analog.
The term ~nucleotide~ refers to a phosphate ester substituted on the 5'-position of a nucleoside. A
nucleotide can have one, two or three phosphoryl groups.
Thus for any given nucleoside, there can be a mono-phosphate, diphosphate and triphosphate ester. Further, the mono-phosphoryl moiety may be linked to two positions of the pentose, forming the 3'5'-cyclic phosphate.
In naming the compounds of the instant invention the following numbering systems will be used for the 0733M (now 07330) 26530-FF

ZC03~08 furanosyl ring:
-CH B

N 3 ~ ~) Y' When the foregoing structure represents a nucleoside, the positions are typically referred to as the prime position (e.g., 4'), whereas the positions on the purine or pyrimidine are not.
Purines are numbered according to the following formula:

~ N \

for e~ample, representing guanine where the 2-position is substituted with NH2 and the 6-position is substituted with -O (named o~o).
Pyrimidines are numbered according to the following formula:

3T~5 2 ~ )6 ~N

for esample, representing thymine where the 2-position is =O
(named oxo), the 4-position is =O (named oxo), and the 5-position is -CH3.
0733M (now 07330) 26530-FF

.

' , , .

20~3408 The position of double bonds in purine and pyrimidine substituen~s will be apparent to those skilled in the art.
It should be further understood that the substitution of a hydrosy or amino on the purine and pyrimidine rin~ also encompasses the tautomeric oxo or imino forms.
The 3',5'-cyclic phosphate esters are represented by the following formula:

HO-P ~

The compounds of the invention will be named using the above-shown numbering system as 4'-substituted nucleosides and derivatives. Some representative compounds are named in the following esamples.
The compound of Formula I where B is thymine, X
is H, ~' is OH, and n is zero, can be named:
4'-azidothymidine, or 1~(4-azido-2-deoxy-~-D-erythro-pentofuranosyl)thymine, or 1-(4-azido-2-deoxy-~-D-erythro-pentofuranosyl)-5-methyl-2,4-diosopyrimidine.
The compound of Formula I where B is 2-amino-purine, X is H, Y' is OH, and n is zero, is named 9-~4-azido-2-deoxy-~-D-erythro- pentofuranosyl)-2-amino-purine.
Certain compounds of the present invention possess asymmetric carbons and may be prepared in either optically activs form, including the ~-D or the ~-L
forms, or as a racemic mixture. Unless otherwise specified, the compounds described herein are all in the ~-D-furanosyl configuration. However, the scope of the subject invention herein is not to be considered limited 0733M (now 07330) 26530-FF

Z0~ 8 to this form, but to encompass all other individual optical isomers of the subject compounds and mixtures thereof.
A chemical bond indicated as (~) refers to the nonspecific stereochemistry of the asymmetric carbon atoms, e.g. at position 4' of the furanosyl ring (see Reaction Scheme B).
~ Pharmaceutically acceptable esters~ and ~ethers~
include those compounds of Formula I where an o~ygen or a nitrogen has been modified, e.g., acylated by the addition of the group -C(=O)-W, wherein W is an alkyl group containing 1 to 20 carbon atoms, adamantyl, aryl, amino, alkylamino, dialkylamino, an alkosy group containing 1 to 20 carbon atoms, -CH2-O-CH3, -CH2-NH2, or a group of the formula:

~N-CH2-A

where A is hydrogen, lower alkyl or aryl ~such compounds prepared in accordance with the teachings of N. ~odor, et al., Pharmac. Ther., 12, 337-386 (1983), where enhanced blood/brain barrier permeability is suggested for compounds having the subject moiety]. Particularly preferred esters are the adamantoate, the palmitoate and the dihydropyridyl esters. This invention contemplates those compounds of Formula I which are esters as described herein and at the same time are the pharmaceutically acceptable acid addi~ion salts thereof.
The invention also contemplates the isopropyl and benzyl ethers of the compounds of Formula I.
A ~pharmaceutically acceptable salt~ may be any salt derived from an inorganic or organic acid or base. The term ~pharmaceutically acceptable anion~ refers to the anion of such acid addition salts. The term 0733M ~now 07330) 26530-FF

ZC~)34~)8 "pharmaceutically acceptable cation~ refers to the cation of such base addition salts. The salt, anion and/or the cation are chosen not to be biologically or otherwise undesirable.
The anions are derived from inorganic acids, such as hydrochloric acid, hydrobromic acid, sulfuric acid (giving the sulfate and bisulfate salts), nitric acid, phosphoric acid and the like, and organic acids such as acetic acid, propionic acid, glycolic acid, pyru~ic acid, oxalic acid, malic acid, malonic acid, succinic acid, maleic acid, fumaric acid, tartaric acid, citric acid, benzoic acid, cinnamic acid, mandelic acid, methanesulfonic acid, ethanesulfonic acid, salicylic acid, p-toluensulfonic acid and the like.
The cations are derived from bases, such as alkaline earth hydro~ides, including calcium hydroxide, potassium hydroxide, sodium hydroside, lithium hydroside and the like, preferably sodium hydroxide.
As used herein, the terms ~inert organic solvent~ or ~inert solvent~ mean a solvent inert under the conditions of the reaction being described in conjunction therewith [including, for example, benzene, toluene, acetonitrile, tetrahydrofuran ~THF~), dimethylformamide ~D~F~), chloroform, methylene chloride (or dichloromethane), diethyl ether, pyridine and the like]. Unless specified to the contrary, the solvents used in the reactions of the present invention are inert organic solvents.
As used herein, the term ~q.s. n means adding a quantity sufficient to achieve a stated function, e.g., to bring a solution to a desired volume ~e.g., 100 mL).
Unless specified to the contrary, the reactions described herein ta~e place at atmospheric pressure over a temperature range from about -20C to about 100C, more preferably from about 10C to about 50C, and most 0733M (now 07330) 2b530-FF

20~)~3408 preferably at about room (or ~ambient~) temperature, e.g., about 20C.
Isolation and purification of the compounds and intermediates described herein can be effected, if desired, by any suitable separation or purification procedure such as, for esample, filtration, estraction, crystallization, column chromatoqraphy, thin-layer chromatography or thick-layer chromatography, or a combination of these procedures. Specific illustrations of suitable separation and isolation procedures can be had by reference to the e~amples hereinbelow. However, other equivalent separation or isolation procedures can, of course, also be used.
As used herein, the term ~treatment~ or ~treating~
means any treatment of a disease in a mammal, including:
(i) preventing the disease, that is, avoiding any clinical symptoms of the disease; -(ii) inhibiting the disease, that is, arresting the development or progression of clinical symptoms; and/or ~ iii) relieving the disease, that is, causing the regre~io~ of clinical symptoms.
As used herein, the term ~effective amount~ means a dosage sufficient to provide treatment for the disease 8tate being treated. This will vary depending on the patient, the disease and the treatment being effected.

SYnthesis of the ComDounds of Formula I

As used in the Reaction Schemes, the substituents B, B', R, X, Y', Z, and n are the same as described in the Summary of the Invention.
The compounds of Formula I where Y' is OH are prepared as described with reference to Reaction Scheme A.

0733M (now 07330) 26530-FF

., : ` -, ~ 20~3~08 Reaction Scheme A

I-C~

OH OH

~2) ~> N2C5 ~3J

OH

I-C~ J

N3~---C~N' NO-C~, t)-R OH
Formula II Formula I-A

0733M (now 07330) 26530-FF

~ "
20~)3408 Startina Materials Referring to Reaction Scheme A, the starting materials of Formula 1 are 2'-deo~y-erythro-pentofuranosyl nucleosides, ribofuranosyl nucleosides and arabino-furanosyl nucleosides selected from the compounds where B
is, for esample, adenine (6-aminopurine), guanine (2-amino-6-o~opurine), uracil (2,4-diosopyrimidine), thymine (5-methyl-2,4-diosopyrimidine), cytosine (4-amino-2-oso-pyrimidine), hypo~anthine (6-hydrosypurine), santhine (2,6-dihydrosypurine), 5-methylcytosine, 4-etho~y-5-methyl-2-oso-pyrimidine, 4-isopropo~y-5-methyl-2-o~o-pyrimidine, and 5-methyl-2-oso-pyrimidine. Many of these materials are available commercially from such suppliers as, Aldrich Chemical Company or Sigma Chemical Company; and where not, they can be easily prepared according to procedures that are well known to the art and published in the literature.

II~E~Ua5iQDL9f Intermediate 2 Referring to Reaction 8cheme A, a compound of Formula 1 i~ iodinated using a misture of triphenylpho8phine, iodine and pyridine, or similar iodinating reagents such as methyltriphenoxyphosphonium iotide, in a solvent (such as diosane, tetrahydrofuran, or dichloromethane). After keeping the misture at a temperature between room temperature and about 50C, preferably at about room temperature, for a period from about 4 hours to 16 hours, preferably about 8 hours; an intermediate of Formula 2 is isolated by evaporation of the 801vents and estraction of the residue, followed by either crystallization or chromatography.

Preparation of Intermediate 3 A compound of Formula 2 is dissolved or suspended in 0733N (now 07330) 26530-FF

20~3408 a solvent (such as methanol or other alcohol, or dioxane, tetrahydrofuran, dimethylformamide, dimethylsulfo~ide;
preferably methanol) by the addition of a base (such as sodium methoxide, potassium t-buto~ide or the like;
preferably sodium methoside). The solution is heated at a temperature from about 50C to about 100C, preferably about 65C; for a period of about 12 hours to 24 hours, preferably about 16 hours. After neutralizing with an acid (such as acetic acid), the solvents are removed by evaporation and a compound of Formula 3 is crystallized out of the residue or isolated by chromatography.
Alternatively, a compound of Formula 3 can be prepared by dissolving compound of Formula 2 in a solvent (such as dimethylformamide, dimethylsulfoxide, N-methyl-2-pyrrolidone or the like) and adding a base (such as 1,8-diazabicyclo~5.4.0]undec-7-ene or 1,5-diazabicyclo-[4.3.0]non-5-ene) and keeping the mixture at about room temperature for a period of about 12 hours to 24 hours, preferably about 16 hours. After removal of the solvent by evaporation, the compound of Formula 3 is purified by chromatography.

~L~a~ation ~ Lntermediate 4 A compound of Formula 4 is prepared by adding a solution of compound of Formula 3 (in dimethylformamide or N-methyl-2-pyrrolidone or the like; preferably DMF) to a miYture of an alkaline metal azide ~such as sodium or lithium azide, preferably sodium azide) and an iodinating agent (such as iodine monochloride or iodine, preferably iodine monochloride) in solution preferably with the same solvant. After stirring the misture for a period of about 5 minutes to 2 hours, preferably about 1 hour; at a temperature range of about 0C to 50C, preferably at about room temperature; the compound is isolated by 0733M (now 07330) 26530-FF

~` 2C~408 estraction, optionally followed by chromatography or by crystallization.

Pre~aration of Com~ounds of Formula_11 A solution of a compound of Formula 4 and an acid chloride or anhydride (such as benzoyl chloride, anisoyl chloride, acetic anhydride, or the like) in a solvent (such as pyridine) is kept at a temperature range of 20C
to 50C, preferably at about room temperature; for a period of 6 hours to 24 hours, preferably for about 16 hours. The compound of Formula II is recovered by evaporation of the solvent and purified by chromatography or by crystallization.

PreDaration of Com~ounds of Formula I
A compound of Formula II is dissolved or suspended in a solvent such as dichloromethane saturated with water and treated with an o~idizing agent (such as a carbo~ylic pero~y acid, e.g., perosybenzoic acid, peracetic acid, 3-chloropero~ybenzoic acid or the like). The reaction by-product8 are removed by e~traction and the residue is treated with a base (such as, aqueous sodium hydro~ide, soaium metho~ide, methanolic sodium metho~ide, methanolic ammonia, ammonium hydroside, agueous dimethylamine or the like). The compound of Formula I-A is isolated by evaporation of the solvent and purification, for inætance, by chromatography.
Compounds of Formula I-A prepared by the above-described pr~cess of the invention may be identified by the presence of a detectable amount of Formula II or the salt or the acid derived from the perosy acid used in the reaction. While it is well known that pharmaceuticals must meet pharmacopoeia standards before approval and/or marketing, and that precursors ~such as Formula II) or side products (such as the acids or salts) should not 0733M (now 07330) 26~30-FF

, .. ~. .

2CC~3~08 esceed the limits prescribed by pharmacopoeia standards, final compounds prepared by a process of the present invention may have minor, but detectable, amounts of such materials present, for esample at levels in the range of 1 part per million to two percent. These levels can be detected, e.g., by HPLC-mass spectrometry. It is important to monitor the purity of pharmaceutical compounds for the presence of such materials, whose presence is additionally disclosed as a method of detecting use of a process of the invention.

The compounds of Formula III, particularly where Y' is OH, are prepared as described with reference to Reaction Scheme B.

0733M (now 07330) 26530-FF

' , Z~ 1C)8 Reaction Scheme B

HO-CH2 B R2-Se-CH2 B
xJ > ~
Y' y~

`Se-CH B
(6) ~2 ~

B B
~7) - > ~ C= < ~J + ~
~, i. I
Y' Y~

R3-o B

(8) >

Y' Formula III
~artina Materials Referring to Reaction Scheme B, the starting materials of Formula 5 are 2'-deoxy-erythro-pentofuran~syl 0733M (now 07330) 26530-FF

20~408 nucleosides, ribofuranosyl nucleosides and arabino-furanosyl nucleosides selected from the compounds where B
is, for example, guanine, adenine, thymine, uracil, cytosine, hypo~anthine, xanthine, 5-methylcytosine, 4-ethosy-5-methyl-2-oso-pyrimidine, 4-isopropo~y-5-methyl-2-oxo-pyrimidine, or 5-methyl-2-oxo-pyrimidin2.
Many of these materials are available commercially from such suppliers as, Aldrich Chemical Company or Sigma Chemical Company; and where not, they can be easily prepared according to procedures that are well known to the art and published in the literature. For esample, see Svnthetic Procedures in Nucleic Acid Ch~mis~r~, Vol.
1, Zorbach and Tipson, Eds., Wiley Interscience (1968);
and Nucleic Acid ChemistrY, parts 1-3, Townsend and Tipson, Eds., Wiley Interscience (1978, 1978, 1986).

Prearation of Intermediate 6 A compound of Formula 5 is reacted with a selenating agent tsuch as, an aryl sele~yl halide or selenocyanate;
preferably o-nitrophenylselenocyanate) in the presence of a pho8phine (e.g. triphenylphosphine, tri-n-butyl-phosphine) in a solvent (such as tetrahydrofuran, dichloromethane, or diosane). After keeping the reaction at a temperature from about 0C to 50C, preferably at about room temperature for a period of about 30 minutes to about 24 hours, preferably about 1 hour, the compound of Formula 6 is isolated following extraction, evaporation and purification, e.g. chromatography.

PreDaration of Intermediate 7 A compound of Formula 6 is dissolved or suspended in a solvent (such as dichloromethane, chloroform or the like) and treated with an oxidizing agent (such as hydrogen peroside or a carbosylic peracid, e.g., perosybenzoic acid, 3-chloroperosybenzoic acid, peracetic 0733M (now 07330) 26530-FF

20~)3~)8 acid or the like). Optionally a neutralizing agent (such as sodium bicarbonate or the like) may be added. The reaction mixture is stirred at a temperature between about 0C and 50C, preferably at about room temperature, for a period of about 5 minutes to about 24 hours, preferably for 30 minutes. The compound of Formula 7 is isolated following extraction and evaporation followed optionally by purification on silica gel or the like.
Preferably, the compound of Formula 7 is converted directly to a compound of Formula 8 without purification.

Pre~aration of Inte~mediates 8 and 9 A compound of Formula 7 is heated in a solvent (such as toluene, benzene or the like) in the presence of a base (such as a trialkyl or triarylamine, preferably triethylamine) to a temperature in the range of about 50C to 150C for a period of about 10 minutes to about 5 bour~, preferably about 1 hour. The compound of Formula 8 is isolated following evaporation and purification and optionally crystallization. Depending on the nature of the substit~ent Y', the isomeric compound of Formula 9 may also be isolated from the reaction misture.

Pre~aration of Comsounds of Formula III
A compound of Formula 8 is reacted with a peracid (such as perosybenzoic acid, 3-chloroperoxybenzoic acid, peracet;c acid or the like) in the presence of an alcohol solvent (such as, a primary lower alkyl alcohol, preferabiy methanol, ethanol or the like). The solution is stirred at a temperature between about -40C and 50C, for a period of a few minutes to about 48 hours, depending on the Y' group. For instance, if Y' is H then the solution is stirred preferably at a temperature between -10C and 0C for about 1 to 2 minutes; if Y' is F then the solution is stirred preferably at about room 0733M (now 0733~ 26530-FF

2c~:)3~a08 temperature for about 1 hour to about 48 hours.
Compounds of Formula III (where R3 is a lower alkyl group corresponding to the primary lower alkyl alcohol used in the reaction) are isolated following e~traction, evaporation and crystallization; optionally purification by chromatography may be employed.
Other 4'-azido compounds of Formula I can be prepared ætarting from the 4~-methosy compounds (for e~ample, the compounds of Formula III made as shown in Reaction Scheme B) as described with reference to Reaction Scheme D.
Reaction Scheme D

Formula III ~ 4 k x) Y ' Formula III or (13) N
Y' Formula I-D
pFeparation of Intermediate 13 A compound of Formula III is treated with a silylating reagent (e.g., a tri-substituted silyl chloride, such as trimethylsilyl chloride, t-butyl-dimethylsilyl chloride or the like) in a solvent (such as dichloromethane, chloroform, tetrahydrofuran, 0733M (now 07330) 26530-FF

.

20~)~408 dimethylformamide, dio~ane or the like); and in the presence of a base (such as triethylamine, pyridine, imidazole or the like); at a temperature in the range of about 0C to 60C, preferably at room temperature, for a period from about 10 minutes to about 10 hours, preferably for about 3 hours. The compound Formula 13 (where R3 is lower al~yl, and any Y' that was OH in Formula III is optionally R4, and R4 is a trisubstituted silylo~y group) is isolated by evaporation, estraction and optionally chromatography.

P~eParation of Formula I-D
Compounds of Formula III or Formula 13 are converted to a compound Formula I-D by dissolving a compound of Formula III or Formula 13 in a solvent (such as dichloromethane, chloroform or the like) and treating it with an azide (such as, azidotrimethylsilane, sodium azide, lithium azide~ or the like) in the presence of a Lewi~ acid catalyst (such a~ trimethylsilyl trifluoro-methane~ulfonate, triisopropylsilyl trifluoromethane-sulfonate, or the like) at a temperature in the range of about 0C to about 100C, preferably at room temperature;
for a period of 10 minutes to 100 hours, preferably 24 to 50 hours. The compound of Formula I is isolated by evaporation, optionally treating it with an aqueous base or fluoride. Finally, optional e~traction is followed by purfication such as silica gel chromatography and crystallization.

PreDaration of the PhosDhonate salts of Formula I
The phosphonate salts of Formula I are prepared by reaction of a compound of Formula II with phosphonous acid or a salt of a di-lower alkyl phosphonate using methods ~nown in the literature.

0733M ~now 07330) 26530-FF

2C~ 08 FreDaration of the PhosPhate Salts of Formula I
Phosphorylating agents useful for preparation of the phosphate salts include, for esample, phosphoryl chloride, pyrophosphoryl chloride and the like, as will be known to those skilled in the art.
The 5~-monophosphate esters of the nucleosides described herein are prepared starting from the parent nucleoside, for esample, using methods described by Imai et al., Journal of Or~anic ChemistrY, ~, 1547 (1969).
The 5'-diphosphate esters and 5'-triphosphate esters of the nucleosides described herein are prepared starting from the monophosphates, for esample, using methods described by Hoard et al., Journal of the American Chemical SocietY,-~, 1785 ~1965~.
The 3',5'-cyclicphoæphate esters of the nucleosides described herein are prepsred starting from the monophosphate~, for esample, using methods described in Smith et al., ~ournal of the American Chemical SocietY, ~, 698 (1961).

PreDaration o the Salts of Formula I
The pharmaceutically acceptable salts of Formula I
are prepared by dissolving a compound of Formula I in a suitable solvent (such as water) adding one to three molar equivalents ~preferably one molar equivalent) of an appropriate acid ~such as hydrochloric acid) or base ~such as an alkaline earth hydroside, e.g., lithium hydro~ide, calcium hydroside, potassium hydro~ide, sodium hydroside or the like; preferably sodium hydroxide) and stirring. The salt is isolated by lyophilization or by precipitation, using techniques that will be apparent to those skilled in the art.

Pre~_ration of the Esters of Formula I
The pharmaceutically acceptable esters of Formula I
0733M (now 07330) 26530-FF

. .
.

20~)3408 are prepared by adding a compound of Formula I and a catalyst (such as 4-dimethylaminopyridine) in pyridine, dropwise to an appropriate acid chloride of the acyl group to be added (suc~ as adamantanecarboxylic acid chloride, palmitic acid chloride, N-methyl-dihydro pyrid-3-ylcarbosylic acid chloride or isopropyl acid chloride) either neat or in a solvent ~such as methylene chloride, dichloroethane or the like~. The reactants are stirred at room temperature for 10 to 24 hours, preferably from 12 to 18 hours. The product is isolated by methods well known in the art such as chromatography.

Preferred Processes and Last Ste~s The compounds of the present invention can be prepared according to the following last steps (in which non-essential substituents are not discussed, but, will be apparent to those skilled in the art from reference to the foregoing reaction schemes):
a 4'-azido-5'-deosy-5'-iodonucleoside is contacted with an acyl halide to give a compound according to Formula II;
a 3'-O-acyl-4'-azido-2',5'-dideoxy-5'-iodonucleoside or a 2',3'-di-O-acyl-4^-azido-5'-deoxy-5'-iodonucleoside i9 contacted with a peracid followed by a base to give a compound according to Formula I;
a compound of Formula III where Z' is methoxy is contacted with an azide in the presence of a Lewis acid catalyst to give a compound according to Formula I;
a compound of Formula I where n is zero is contacted with a phosphorylating agent to give a compound according to Formula I where n is one;
a compound of Formula I where n is one is contacted with a phosphorylating agent to give a compound according to Formula I where n is three;
a compound of Formula I where n is one is contacted 0733M (now 07330) 26530-FF

with a cyclizing agent (such as dicyclohe~ylcarbodiimide~
to give a compound according to Formula I where Y' and Z
together form a cyclic phosphate ester;
a compound of formula II is reacted with phosphonous acid or a salt of a di-lower alkyl phosphonous acid to give a compound of formula I wherein Z is (R'0)2P(O)CH2-;
a compound of formula I, following protection of reactive nitrogen atoms on the purine or pyrimidine heterocycle by acylation (e.g., benzoylation), is reacted with a strong base (e.g., sodium hydride) followed by addition of an alkyl halide (such as benzyl bromide or 2-iodopropane) to give the corresponding ether compound of Formula I after deacylation;
a compound of Formula I is contacted with a pyridine catalyst and an acid chloride to give the corresponding ester;
a compound of Formula I is contacted with a pharmaceutically acceptable base to form the corresponding base addition salt of Formula I;
substituting a pharmaceutically acceptable base addition salt of Formula I with another pharmaceutically acceptable base; and contacting an base addition salt of Formula I with a acid to form the corresponding free acid compound of Formula I.

Preferred Com~ounds Presently preferred are the compounds of Formula I
where B is guanine, adenine, thymine, uracil, cytosine, hypoxanthine, xanthine, 5-methylcytosine, 4-ethoxy-5-methyl-2-oxo-pyrimidine, 4-isopropoxy-5-methyl-2-oxo-pyrimidine, or 5-methyl-2-oxo-pyrimidine; especially the compounds where B is adenine, guanine, uracil, thymine, or cytosine.
0733M (now 07330) 2~530-FF

. ' ' '' , ' . .
' ' -, .

Also preferred are the compounds of Formula I where iS H;
particularly the compounds where Y' is OH and B iS thymine, uracil, cytosine, guanine or adenine;
and also the compound where Y' is H and B is thymine.
Still other preferred compounds of Formula I are those where B is thymine.
Most preferred is the compound 4'-azidothymidine.

UtilitY. Testina and Administration General UtilitY
The compounds of this invention are particularly useful for treating viral, bacterial and fungal infections.
Generally, the infections treated with the compounds o~ the present invention are found in mammals, including:
animals ~uch as mice, monkeys and the like; and particularly humans.
The oompounds of the present invention, including the pharmaceutically acceptable salts and esters thereof, and the compositions containing them are useful as potent antiviral agents, particularly against human immunodeficiency virus (HIV).

Testina In vitro testing for antiviral activity against HIV
is done, for esample, by the procedures described by Chen et al., Biochemical PharmacoloaY, 36 (24), 4361-4362 (1987), or modifications thereof.
Inhibition of reverse transcriptase and human polymerase is determined by the procedures described by Chen et al., ~olecular Pharmacoloav, 25, 441-445 (1984), or as described by Wang et al., Biochemistry, 21, 0733M (now 07330~ 26530--FF

1597-1608 (1982), or by modifications thereof.
Tests for to~icity can be carried out by the procedures described by Diainiak, et al., British Journal of HaematoloaY, 69, 229-304 (1988), or as described by Sommadossi, et al., Aaents and ChemotheraDY, 31 (3), 452-454 (1987), or by modifications thereof.
In vivo testing to demonstrate the described antiviral activity of the present compounds is done, for example, by procedures described by Jones et al., Journal of ViroloaY, 62 (2), 511-518 (1988), or by modifications thereof.

Administration The compounds of this invention are administered at a therapeutically effective dosage, i.e., a dosage sufficient to provide treatment for the disease states previously described. Administration of the active compound8 and salts described herein can be via any of the accepted modes of administration for agents that serve similar utilities.
Generally, an acceptable daily dose is of about 0.01 to 150 mg per kilogram body weight of the recipient per day, preferably about 1.5 to 75 mg per kilogram body weight per day, and most preferably about 5 to 30 mg per kilogram body weight per day. Thus, for administration to a 70 kg person, the dosage range would be about 0.7 mg to 10.5 g per day, preferably about 105 mg to 5.25 g per day, most preferably 350 mg to 2.0 g per day.
Administration can be via any accepted systemic or local route, for example, via parenteral, oral (particularly for infant formulations), intravenous, nasal, transdermal or topical routes, in the form of solid, semi-solid or liquid dosage forms, such as for example, tablets, suppositories, pills, capsules, powders, solutions, suspensions, aerosols, emulsions or 0733M (now 07330) 26530-FF

20~3408 the like, preferably in unit dosage forms suitable for simple administration of precise dosages. The compositions will include a conventional pharmaceutical carrier or e~cipient and an active compound of Formula I
and, in addition, may include other medicinal agents, pharmaceutical agents, carriers, adjuvants, etc.
For example, in methods of treating AIDS infections, particularly where ths compromised subject is suffering from other viral infections, such as, herpes, an active compound of Formula I can be co-administered with one or more agents active in reducing viral infections, such as, acyclovir, ganciclovir, and foscarnet which have been demonstrated to reduce the severity of herpetic viral infections. Co-administration can be in the form of a single formulation (combining, for example, a compound of Formula I and ganciclovir with pharmaceutically acceptable excipients, optionally segregating the two active ingredients in different escipient mixtures designed to independently control their respective release rates and durations) or by independent administration of separate formulationæ containing the active agents.
If desired, the pharmaceutical compoæition to be administered may also contain minor amounts of non-to~ic ausiliary substances such as wetting or emulsifying agents, pH buffering agents and the liXe, such as for esample, sodium acetate, sorbitan monolaurate, triethanolamine oleate, etc.
The compounds of this invention are generally administered as a pharmaceutical composition which comprises a pharmaceutical e~cipient in combination with a compound of Formula I. The level of the drug in a formulation can vary within the full range employed by those skilled in the art, e.g., from about 0.01 percent weight (%w) to about 99.99%w of the drug based on the 0733M (now 07330) 26530-FF

20~3~08 total formulation and about 0.01%w to 99.99%w e~cipient.
Preferably, the formulation will be about 3.5 to 60% by weight of the pharmaceutically active compound, with the rest being suitable pharmaceutical excipients.

Intravenous Administration Intravenous injection has proven to be an important route of administration for antiviral agents. The compounds of the present invention can be administered via this route, for example, by dissolving the compound, salt, ester or ether in a suitable solvent ~such as water or saline) or incorporation in a liposomal formulation followed, by dispersal into an acceptable infusion fluid. A typical daily dose of a compound of the invention can be administered by one inusion, or by a serie8 of infusions spaced over periodic intervals.

QLal Administration Oral administration can be used to deliver the compound of Formula I using a convenient dàily doæage regimen which can be adjusted according to the degree of affliction or for renal impairment, or to compensate for the tosic effects of other medications administered contemporaneously. For such oral administration, a pharmaceutically acceptable, non-toxic composition is formed by the incorporation of any of the normally employed escipients, such as, for example, pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharine, talcum, cellulose, glucose, gelatin, sucrose, magnesium carbonate, and the like. Such compositions take the form of solutions, suspensions, tablets, pills, capsules, powders, sustained release formulations and the like. Such compositions may coDtain between 0.01 wt/wt% and 99.99 wt/wt% of the compound of Formula I, but preferably such compositions will contain 0733M (now 07330) 26530-FF

- 2C~408 between 25 wt/wt~ and about 80 wt/wt%.
Preferably the compositions will take the form of a capsule, pill or tablet and thus the composition will contain, along with the active ingredient, a diluent such as lactose, sucrose, dicalcium phosphate, and the like; a disintegrant such as starch or derivatives thereof; a lubricant such as magnesium stearate and the like; and a binder such as a starch, polyvinylpyrrolidone, gum acacia, gelatin, cellulose and derivatives thereof, and the like. For oral administration to infants, a liquid formulation (such as a syrup or suspension) is preferred.

LiDosomal Formulations Pharmaceutical formulations based on liposomes have recently reached human clinical trials. Their benefits are believed related to favorable changes in tissue distribution and pharmacokinetic parameters that result from liposome entrapment of drugs, and may be applied to the compounds of the present invention by those skilled in the art.
The formulations can be designed to either target drug to disease sites tsee: Lopez-Berestein et al., J. Infect. Dis., 1~1: 704-710 (1985); Gotfredsen et al., ~iochemical PharmacoloaY, 32: 3389-3396 (1983)]; or to the reticuloendothelial system tsee Eppstein et al., Int. 3. Immunothera~Y, 2: 115-126 (1986)], to increase duration of drug action [see: Gabizon et al., Cancer 4734 (1982); Eppstein et al., DeliverY SYstems for PeDtide Druas, Eds. S.S. Davis, L. Illum and E.
Tomlinson, Plenum Pub. Corp., New Yor~, pp. 277-283; C.A.
Hunt, Biochemica et BioPhYsica Acta., 719: 450-463 (1982); and Senior et al., Biochemica et ~ioPhYsica Acta., 839: 1-8 (1985)], or to divert a drug away from organs that are particularly sensitive to its toxic effects [see: Weinstein et al., Pharmac. Ther., 24:
0733M (now 07330) 26530-FF

2003~08 207-233 (1983); Olson et al., Eur. J. Cancer Clin.
Oncol., 18: 167-176 (1982); and Gabzion et al., suDra.].
Controlled release liposomal liquid pharmaceutical formulations for injection or oral administration are described in U.S. Patent No. 4,016,100. Liposomal applications for oral drug delivery of a lyophilized liposome~peptide drug misture filled into intestine capsules have also been suggested, see U.S. Patent No.
4,348,384. Additionally, viral infections of the eye (such as herpetic keratitis and HIV retinitis) may be treated by use of a æustained release drug delivery system as described in U.S. Patent No. 4,217,898.

SuD~ositories For systemic administration via suppository, traditional binders and carriers include, for e~ample, polyalkaline glycol or triglycerides [e.g., PEG 1000 (96%) and PEG 4000 (4%)1. Such suppositories may be formed from mi~tures containing active ingredients in the range of rom about 0.5 wt/wt% to about 10 wt/wt%;
preferably from about 1 wt/wt~ to about 2 wt/wt%.

~iquids Liquid pharmaceutically administerable compositions can, for e~ample, be prepared by dissolving, dispersing, etc. an active compound (about 0.5~ to about 20%), as described above, and optional pharmaceutical adjuvants in a carrier, such as, for e~ample, water, saline, aqueous destrose, glycerol, ethanol and the like, to thereby form a solution or suspension.
Actual methods of preparing such dosage forms are known, or will be apparent, to those skilled in this art:
for esample, see Reminaton's Pharmaceutical Sciences, Mack Publishing Company, Easton, Pennsylvania, 16th Ed., 1980. The composition to be administered will, in any 0733M ~now 07330) 26530-FF

, , -200~408 event, contain a quantity of the active compound(s) in a pharmaceutically effective amount for relief of the particular condition being treated in accordance with the teachings of this invention.

EXAMPLES
The following preparations and esamples are given to enable those skilled in the art to more clearly understand and to practice the present invention. They should not be considered as limiting the scope of the invention, but merely as being illustrative and representative thereof.
Pre~aration 1 lA. Formula 2 Where B is Thvmine. X is H
To a suspension of thymidine (0.968 g, 4.0 mM) in diosane ~20 mL) containing pyridine (0.65 mL, 8.0 mM) was added triphenylpho~phine (1.57 g, 6.0 mM) and iodine ~1.52 g, 6.0 mM). After stirring the misture for 7 hours at 21C, methanol (1.0 mL) was added, followed by removal ~f the solvent by evaporation. A solution of the residue in ethyl acetate (150 mL) was estracted successively with water (30 mL), 10% aqueous sodium thiosulfate solution (30 m~), and brine (30 mL). The ethyl acetate phase was concentrated in vacuo to a syrup which was taken up in hot ethanol (50 m~) and filtered. The filtrate was concentrated to a volume of 15 m~. On cooling, 5'-deo~y-5'-iodothymidine crystallized out. The crystals were filtered, rinsed with ethyl acetate, and dried, affording 5'-deoxy-5'-iodothymidine (0.806 g), a compound according to Formula 2.

lB. Formula 2 VarYina B
By following the procedure of part A and substituting 0733M (now 07330) 26530-FF

ZC~408 for thymidine with the following:
2'-deosyadenosine, 2'-deosyguanosine, 2'-deosyuridine, 2'-deo~ycytidine, 1-(2-deosy-~-D-erythro-pentofuranosyl)-4-amino-5-methyl-2-osopyrimidine, 1-(2-deosy-~-D-erythro-pentofuranosyl)-4-ethosy-S-methyl-2-osopyrimidine, 1-(2-deosy-~-D-erythro-pentofuranosyl)-4-iso-proposy-S-methyl-2-osopyrimidine, 1-(2-deosy-~-D-erythro-pentofuranosyl)-5-methyl-2-osopyrimidine, 9-(2-deosy-~-D-erythro-pentofuranosyl)-6-hydrosy-purine, and 9-(2-deosy-~-D-erythro-pentofuranosyl)-2,6-di-hydrosypurine;

there are obtained the following respective compounds:
2',5'-diaeosy-S'-iodoadenosine, 2',5'-dideosy-S'-iodoguanosine, 2',5'-dideosy-5'-iodouridine, 2',5'-dideosy-S'-iodocytidine, 1-(2,5-dideosy-5-iodo-~-D-erythro-pentofuranosyl)-4-amino-5-methyl-2-osopyrimidine, 1-(2,5-dideosy-S-iodo-~-D-erythro-pentofuranosyl)-4-ethosy-5-methyl-2-osopyrimidine, 1-(2,5-dideosy-5-iodo-~-D-erythro-pentofuranosyl)-4-isoproposy-5-methyl-2-osopyrimidine, 1-(2,5-dideo~y-5-iodo-~-D-erythro-pento~uranosyl)-S-methyl-2-osopyrimidine, 9-(2,5-dideosy-S-iodo-~-D-erythro-pentofuranosyl)-6-hydrosypurine, and 9-(2,5-dideosy-5-iodo-~-D-erythro-pentofuranosyl)-2,6-dihydrosypurine.
0733M (now 07330) 26530-FF

.

.
.
.
.

ZC~ 8 PreearatiQn 2 2A. Formula 3 Where B is Thvmin~ X is H

A lN solution of sodium methoxide in methanol (0.85 mL) was added to a suspension of 5'-deoxy-5'-iodothymidine (100 mg, 0.284 mM), prepared, e.g., as described in Preparation lA, in anhydrous methanol (5 m~). The solution was heated at reflus for 16 hours, cooled to room temperature, and neutralized by the addition of glacial acetic acid. After removal of solvents by evaporation, the residue was crystallized from ethanol to give 58 mg of 5'-deoxythymidin-4'-ene (a compound according to Formula 3) (mp 208-210C).

2B. Formula 3 varvina B
~ y following the procedure of part A and substituting for 5'-deosy-5~-iodothymidine the following:
2~,5'-dideosy-5'-iodoadenosine, 2~,5'-dideosy-5'-iodoguanosine, 2',5'-dideosy-5'-iodouridine, 2',5'-dideosy-5'-iodocytidine, 1-(2,5-dideoxy-5-iodo-~-D-erythro-pentofuranosyl)-4-amino-5-methyl-2-osopyrimidine, 1-~2,5-dideosy-5-iodo-~-D-erythro-pentofuranosyl)-4-ethosy-5-methyl-2-osopyrimidine, 1-(2,5-dideoxy-5-iodo-~-D-erythro-pentofuranosyl)-4-isoproposy-5-methyl-2-o~opyrimidine, 1-(2,5-dideosy-5-iodo-~-D-erythro-pentofuranosyl)-5-methyl-2-oxopyrimidine, 9-(2,5-dideoxy-5-iodo-~-D-erythro-pentofuranosyl)-6-hydrosypurine, and 9-(2,5-dideosy-5-iodo-~-D-erythro-pentofuranosyl)-2,6-dihydrosypurine;

0733M (now 07330) 26530-FF

, , 20g)3408 there are obtained the following respective compounds:
2'5'-dideo~yadenosin-4'-ene, 2'5'-dideosyguanosin-4'-ene, 2'5'-dideosyuridin-4'-ene, 2'5'-dideosycytidin-4'-ene, 1-(2,5-dideo~y-~-D-erythro-pent-4-enofuranosyl)-4-amino-5-methyl-2-osopyrimidine;
1-(2,5-dideosy-B-D-erythro-pent-4-enofuranosyl)-4-ethosy-5-methyl-2-o~opyrimidine, 1-(2,5-dideosy-~-D-erythro-pent-4-enofuranosyl)-4-isoproposy-5-methyl-2-osopyrimidine, 1-(2,5-dideosy-~-D-erythro-pent-4-enofuranosyl)-5-methyl-2-o~opyrimidine, 9-(2,5-dideosy-~-D-erythro-pent-4-enofuranosyl)-6-hydrosypurine, and 9-(2,5-dideosy-~-D-erythro-pent-4-enofuranosyl)-2,6-dihydrosypurine.

Pre~aration 3 ~A. Formula 4 Where B is Thvmine. X is H

To a stirred suspension of sodium azide (8.70 g, 134 mM) in dimethylformamide (60 mL), under nitrogen, was added iodine monochloride (10.8 9, 67 mM). After 20 min, a solution of 5'-deosythymidin-4'-ene ~6.00 g, 26.8 mM), prepared, e.g., as described in Preparation 2, in dimethylformamide (600 mL) was added dropwise over 30 min. The misture was allowed to stir at 21C for an additional hour. Saturated aqueous sodium bicarbonate was added (200 mL) followed by enough saturated aqueous sodium thiosulfate solution to render the misture colorle8s. The mi~ture was filtered and the filtrate was evaporated to an oil. A solution of the oil in H2O
~300 mL) was extracted four times with ethyl acetate 0733M ~now 07330) 26530-FF

.

, , . ' ' ' :

-` Z003408 (250 mL portions). The combined organic estracts were dried (MgSO4), and the solvent removed by evaporation to give 4'-azido-5'-deoxy-5'-iodothymidine (a compound according to Formula 4) as 12g of viscous syrup of sufficient purity for subsequent reactions.

3B. Formula 4 VarYina B
By following the procedure of part A and substituting for thymidin-4'-ene the following:
2'5'-dideosyadenosin-4'-ene, 2'5'-dideoxyguanosin-4'-ene, 2'5'-dideoxyuridin-4'-ene, 2'5'-dideoxycytidin-4'-ene, 1-(2,5-dideosy-~-D-erythro-pent-4-enofuranosyl)-4-amino-5-methyl-2-oxopyrimidine;
1-(2,5-dideosy-~-D-erythro-pent-4-enofuranosyl)-4-ethosy-5-methyl-2-osopyrimidine, 1-(2,5-dideo~y-~-D-erythro-pent-4-enofuranosyl)-4-isopropoxy-5-methyl-2-oxopyrimidine, 1-~2,5-dideoxy-~-D-erythro-pent-4-enofuranosyl)-5-methyl-2-oxopyrimidine, 9-~2,5-dideoxy-~-D-erythro-pent-4-enofuranosyl)-6-hydrosypurine, and 9-(2,5-dideoxy-~-D-erythro-pent-4-enofuranosyl)-2,6-dihydroxypurine;

there are obtained the following respective compounds:
4'-azido-2',5'-dideoxy-5'-iodoadenosine, 4'-azido-2',5'-dideoxy-5'-iodoguanosine, 4'-azido-2',5'-dideoxy-5'-iodouridine, 4'-azido-2',5'-dideoxy-5'-iodocytidine, 1-(4-azido-2,5-dideo~y-5-iodo-~-D-erythro-pentofuranosyl)-4-amino-5-methyl-2-oxopyrimidine, 1-(4-azido-2,5-dideoxy-5-iodo-~-D-erythro-pentofuranosyl)-4-ethoxy-5-methyl-2 -oxopyr imidine, 0733M (now 07330) 26530-FF

Z0~3~08 1-(4-azido-2,5-dideoxy-5-iodo-~-D-erythro-pentofuranosyl)-4-isopropoxy-5-methyl-2-oxopyrimidine, 1-(4-azido-2,5-dideosy-5-iodo-~-D-erythro-pentofuranosyl)-5-methyl-2-osopyrimidine, 9-(4-azido-2,5-dideoxy-5-iodo-~-D-erythro-pentofuranosyl)-6-hydroxypurine, and 9-(4-azido-2,5-dideoxy-5-iodo-~-D-erythro-pentofuranosyl)-2,6-dihydroxypurine.

PreDaration 4 4A. Formula 6 Where B is Thymine; Y' is F;
and X is H
3'-Deosy-3~-fluorothymidine (2.04 g, 8.34 mM) and o-nitrophenylselenocyanate (2.10 g, 9.20 mM) were placed in a 1ask under nitrogen. Tetrahydrofuran ~dry) (50 mL) was added followed by 2.2 m~ (8.83 mM) of tri-n-butylphosphine. The misture was stirred at room temperature for 1.5 hours, diluted with 200 mL of ethyl acetate and washed with 200 mL of sa~urated sodium bicarbonate followed by 200 mL of brine. The estract was dried over magnesium sulfate and concentrated under reduced pressure to a yellow oil ~6.3 g).
Chromatographic purification of the yellow oil on silica gel with hesane:ethyl acetate ~4:1 v/v) as eluent gave 2.79g (6.51 mM; 78%) of 3',5'-di-deoxy-3'-fluoro-5'-[(2-nitrophenyl)selenyl]thymidine as an amorphous yellow solid.

4B Formula 6 VarYina B
By following the procedure of part A and substituting 3'-deosy-3'-fluorothymidine with the following:
2',3'-dideoxy-3'-fluoroadenosine, 2',3'-dideoxy-3'-fluorouridine, 0733M (now 07330) 26530-FF

ZC~)3~08 2',3'-dideoxy-3'-fluorocytidine, 2',3'-dideosy-3'-fluoroguanosine, 1-(2,3-dideoxy-3-fluoro-~-D-erythro-pentofuranosyl~-4-amino-5-methyl-2-oxopyrimidine, 1-~2,3-dideosy-3-fluoro-~-D-erythro-pentofuranosyl)-4-ethoxy-5-methyl-2-oxopyrimidine, 1-(2,3-dideosy-3-fluoro-~-D-erythro-pentofuranosyl)-4-isopropoxy-5-methyl-2-osopyrimidine, 1-(2,3-dideoxy-3-fluoro-~-D-erythro-pentofuranosyl~-5-methyl-2-oxopyrimidine, 9-(2,3-dideoxy-3-fluoro-~-D-erythro-pentofuranosyl)-6-hydroxypurine, and 9-(2,3-dideoxy-3-fluoro-~-D-erythro-pentofuranosyl)-2,6-dihydroxypurine;

there are obtained the following respective compounds:
2',3',5'-trideosy-3'-fluoro-5'-t(2-nitrophenyl)-selenyl]adenosine, 2',3',5'-trideoxy-3'-fluoro-5'-t(2-nitrophenyl)-8elenyl]uridine, 2',3',5'-trideoxy-3'-fluoro-5'-t(2-nitrophenyl)-selenyl~cytidine, 2',3',5'-trideoxy-3'-fluoro-5'-[(2-nitrophenyl)-selenyl]guanosine, 1-{2,3,5-trideoxy-3-fluoro-5-~(2-nitrophenyl)-selenyl]-~-D-erythro-pentofuranosyl}-4-amino-5-methyl-2-oxopyrimidine, 1-{2,3,5-trideoxy-3-fluoro-5-t(2-nitrophenyl)-selenyl]-~-D-erythro-pentofuranosyl}-4-ethoxy-S-methyl-2-oxopyrimidine, 1-{2,3,5-trideoxy-3-fluoro-5-~(2-nitrophenyl~-selenyl]-~-D-erythro-pentofuranosyl}-4-isopropoxy-5-methyl-2-oxopyrimidine, 1-{2,3,5-trideoxy-3-fluoro-5-[(2-nitrophenyl)-selenyl]-~-D-erythro-pentofuranosyl}-5-methyl-0733M (now 07330) 26530-FF

2-oxopyrimidine, 9-{2,3,5-trîdeoxy-3-fluoro-5-~(2-nitrophenyl)-selenyl]-~-D-erythro-pentofuranosyl}-6-hydro~ypurine, and 9-{2,3,5-trideo~cy-3-fluoro-5-[(2-nitrophenyl)-selenyl]-~-D-erythro-pentofuranosyl}-2,6-dihydroxy-purine.

Pre~aration 5 5A. Formula 8 Where B is Thymine; Y' is F;
and X is H
3',5'-Dideo~y-3'-fluoro-5'-[(2-nitrophenyl)selenyl]-thymidine (2.79 g, 6.51 mM) was dissolved in 53 mL of methylene chloride. Saturated sodium bicarbonate ~17.8 mL) was added followed under vigorous stirring by 3-chloroperosybenzoic acid (85% grade3~1.37 g, 6.83 mM).
The mi~ture was stirred for 1 hour at room temperature, then poured into 200 mL of 10~ tw/v) sodium thiosulfateJwater. The misture was extracted twice with 200 mL of ld% n-butanol/chloroform. The e~tracts were washed with brine, combined and dried over magnesium sulfate. Concentration under reduced pressure gave 2.90g of selenoxides as a yellow solid.
The crude selenoxides (2.90 g) were suspended in 100 mL of toluene and triethylamine (3.0 mL, 21.5 mM) was added. The mixture was heated to 100C for 1 hour, cooled to room temperature and evaporated at reduced pressure to a semi-solid residue. Chromatography of the residue on silica gel with ethyl acetate:hexane ~4:1 v/v~
as eluent and crystallization from ethyl acetate/hexane gave 650 mg (2.87 mM, 44%) of 3',5'-dideoxy-3'-fluoro-thymidin-4'-ene (mp 167-169C).

0733M (now 07330) 26530-FF

2(~3~08 5B Formula 8 VarYina B
By following the procedure of part A and substituting 3',5'-dideosy-3'-fluoro-5'-[(2-nitrophenyl)selenyl]-thymidine with the following:
2',3',5'-trideo~y-3'-fluoro-5'-~(2-nitrophenyl)-selenyl]adenosine, 2',3',5'-trideoxy-3'-fluoro-5'-t(2-nitrophenyl)-selenyl]uridine, 2',3',5'-trideosy-3'-fluoro-5'-[(2-nitrophenyl)-selenyl]cytidine, 2',3',5'-trideoxy-3'-fluoro-5'-[(2-nitrophenyl)-selenyl]guanosine, 1-{2,3,5-trideoxy-3-fluoro-5-t(2-nitrophenyl)-selenyl]-~-D-erythro-pentofuranosyl}-4-amino-5-methyl-2-oxopyrimidine, 1-{2,3,5-trideosy-3-fluoro-5-t(2-nitrophenyl)-selenyl]-~-D-erythro-pentofuranosyl}-4-ethoxy-5-methyl-2-oxopyrimidine, 1-~2,3,5-trideosy-3-fluoro-5-[(2-nitrophenyl)-selenyl]-~-D-erythro-pentofuranosyl}-4-isopropoxy-5-methyl-2-oxopyrimidine, 1-{2,3,5-trideo~y-3-fluoro-5-t(2-nitrophenyl)-selenyl]-~-D-erythro-pentofuranosyl}-5-methyl-2-osopyrimidine, 9-{2,3,5-trideosy-3-fluoro-5-t(2-nitrophenyl)-selenyl~-~-D-erythro-pentofuranosyl}-6-hydrosypurine, and 9-{2,3,5-trideoxy-3-fluoro-5-t(2-nitrophenyl)-selenyl]-~-D-erythro-pentofuranosyl}-2,6-dihydroxy-purine;

there are obtained the following respective compounds:
2',3',5'-trideoxy-3'-fluoroadenosin-4'-ene, 2',3',5'-trideoxy-3'-fluorouridin-4'-ene 2',3',5'-trideoxy-3'-fluorocytidin-4'-ene, 0733M (now 07330) 26530-FF

Z0~)~408 2',3',5'-trideoxy-3'-fluoroguanosin-4'-ene, l-(2,3,5-trideo~y-3-fluoro-~-D-erythro-pent-4-eno-furanosyl)-4-amino-5-methyl-2-osopyrimidine, 1-(2,3,5-trideosy-3-fluoro-~-D-erythro-pent-4-eno-furanosyl)-4-ethosy-S-methyl-2-o~opyrimidine, 1-(2,3,5-trideosy-3-fluoro-~-D-erythro-pent-4-eno-furanosyl)-4-isopropo~y-S-methyl-2-osopyrimidine, 1-(2,3,5-trideosy-3-fluoro-~-D-erythro-pent-4-eno-furanosyl)-S-methyl-2-osopyrimidine, 9-(2,3,5-trideosy-3-fluoro-~-D-erythro-pent-4-eno-furanosyl)-6-hydrosypurine, and 9-(2,3,5-trideosy-3-fluoro-~-D-erythro-pent-4-eno-furanosyl~-2,6-dihydrosypurine.

~re~aration 6 6A. Formula 6 Where B is Thymine;
and X and Y' are H
3'-Deosythymidine (2.0 g, ~.84 mM) and o-nitro-phenyl~elenocyanate (2.16 9, 9.51 mM) were placed in a flask under nitrogen. Tetrahydrofuran (dry) (50 mL) was added followed by tri-n-butylphosphine (2.4 mL, 9.63 mM).
The misture was stirred at room temperature for l hour, diluted with 250 m~ of ethyl acetate and washed with 250 mL of sat. sodium bicarbonate followed by 250 mL of brine. The organic phase was dried over magnesium sulfate and concentrated under reduced pressure to a yellow oil (6.3 g). Chromatography of the crude oil on silica gel with a hesane:ethyl acetate (1:10 v/v) gave 3',5'-dideosy-5'-[(2-nitrophenyl)selenyl]thymidine (3.15 g, 7.67 mM, 87%) aæ an amorphous yellow solid.
, .
69. Formula 6 VarYina B
By following the procedure of part A and substituting 0733M (now 07330) 26530-FF

, .

- , . ~ . , ` 2003408 3'-deoxythymidine with the following:
2',3'-dideosyadenosine, 2',3'-dideosyuridine, 2',3'-dideosycytidine, 2',3'-dideosyguanosine, 1-(2,3-dideosy-~-D-erythro-pentofuranosyl)-4-amino-5-methyl-2-osopyrimidine, 1-(2,3-dideosy-~-D-erythro-pentofuranosyl)-4-ethosy-5-methyl-2-osopyrimidine, 1-(2,3-dideosy-~-D-erythro-pentofuranosyl~-4-isoproposy-5-methyl-2-osopyrimidine, 1-(2,3-dideosy-~-D-erythro-pentofuranosyl)-5-methyl-2-osopyrimidine, 9-(2,3-dideosy-~-D-erythro-pentofuranosyl)-6-hydrosypurine, and 9-(2,3-dideosy-~-D-erythro-pentofuranosyl)-2,6-dihydrosypurine;

there are obtained the following respective compounds:
5'-1(2-nitrophenyl)selenyl]-2',3',5'-trideosy-aaeno~ine, 5'-t(2-nitroPhenyl)selenyl]-2~3~5~-tride uridine, 5'-1(2-nitrophenyl)selenyl]-2',3~,5'-trideoxy-cytidine, 5'-~(2-nitrophenyl)selenyl]-2',3',5'-trideosy-guanosine, 5-[(2-nitrophenyl)selenyl]-2,3,5-trideosy-~-D-erythro-pentofuranosyl}-4-amino-5-methyl-2-oxo-pyrimidine, 1-{5-1(2-nitrophenyl)selenyl]-2,3,5-trideosy-~-D-erythro-pentofuranosyl}-4-ethoxy-5-methyl-2-oxo-pyrimidine, 1-{5-~(2-nitrophenyl)selenyl]2,3,5-trideoxy-~-D-erythro-pentofuranosyl}-4-isopropoxy-5-methyl-0733M (now 07330) 26530-FF

.

, .. .. ~

2(~ 40~3 2-oxopyrimidine;
1-{5-[(2-nitrophenyl)selenyl]2,3,5-trideosy-~-D-erythro-pentofuranosyl}-5-methyl-2-oxopyrimidine, 9-{5-[(2-nitrophenyl)selenyl]2,3,5-trideosy-~-D-erythro-pentofuranosyl}-6-hydrosypurine, and 9-{5-[(2-nitrophenyl)selenyl]2,3,5-trideosy-,B-D-erythro-pentofuranosyl}-2,6-dihydrosypurine.

Pre~aration 7 7A. E~ormulae 8 and 9 Where B is Thymine;
and X and Y' are H
The 3',5'-dideosy-5'-[(2-nitrophenyl)selenyl]-thymidine (3.04 g, 7.41 ~) was dissolved in 20 mL of methylene chloride. Saturated sodium bicarbonate (20 mL) was added followed under vigorous stirring by 3-chloroperosybenzoic acid (85% grade)(l.58 g, 7.78 mM).
The misture was stirred for 20 min at room temperature, then poured into 20 mL of 10% (v/v) sodium thiosulfate/water. The misture was extracted twice with 200 mL of methylene chloride and three times with chloroform S2S200 mI., lxlO0 mL). The estracts were washed with ~rine, combined and dried over magnesium sulfate. Evaporation of the solvent gave 3.06 g of selenosides as a yellow solid.
The crude selenosides (3.00 g,) were suspended in 100 mL of toluene and triethylamine (3.0 mL, 21.5 n~q) was added. The misture was reflused for 1 hour, cooled to room temperature, and evaporated at reduced pressure to a semi solid residue. Chromatography of the residue on silica gel with ethyl acetate:hesane (6:4 v/v) as eluent, followed by crystallization from ethyl acetate~hesane gave 3',5'-dideoxythymidin-4'-ene 646 mg (44%) (mp 149-153C) and 3',5'-dideoxythymidin-3'-ene (286 mg, 19.5%) (mp 155-156C).
0733M (now 07330) 26530-FF

7B. Formula 8 Varvina B
By following the procedure of part A and substituting for 3',5'-dideosy-5'-[(2-nitrophenyl)selenyl]thymidine with the following:
5'-[(2-nitrophenyl)selenyl]-2',3',5'-trideoxy-adenosine, 5'-[(2-nitrophenyl)selenyl]-2',3',5'-trideoxy-uridine, 5'-tt2-nitrophenyl)selenyl3-2~,3~,5~-trideosy cytidine, 5'-[(2-nitrophenyl)selenyl]-2',3',5'-trideosy-guanosine, 1-~5-t(2-nitrophenyl)selenyl]-2,3,5-trideosy-~-D-erythro-pentofuranosyl}-4-amino-5-methyl-2-oso-pyrimidine, 1-{5-t(2-nitrophenyl)selenyl]-2,3,5-trideosy-~-D-erythro-pentofuranosyl}-4-ethoxy-5-methyl-2-oxo-pyrimidine, 1-{5-~(2-nitrophenyl)selenyl]2,3,5-trideosy-~-D-erythro-pentofuranosyl}-4-isopropoxy-5-methyl-2-osopyrimidine;
1-{5-[~2-nitrophenyl)selenyl]2,3,5-triaeoxy-~-D-erythro-pentofuranosyl}-5-methyl-2-osopyrimidine, 9-{S-~(2-nitrophenyl)selenyl]2,3,5-trideosy-~-D-erythro-pentofuranosyl}-6-hydrosypurine, and 9-{5-~(2-nitrophenyl)selenyl]2,3,5-triaeoxy-~-D-erythro-pentofuranosyl}-2,6-dihydrosypurine;

there are obtained the following respective compounds:
2',3',5'-trideo~yadenosin-4'-ene, 2',3',5'-trideoxyuridin-4'-ene, 2',3',5'-trideosycytidin-4'-ene, 2',3',5'-trideosyguanosin-4'-ene, 1-(2,3,5-trideosy-~-pent-4-enofuranosyl3-4-amino-5-methyl-2-oxopyrimidine, 0733M (now 07330) 26530-FF

.

2C~)3408 1-(2,3,5-trideoxy-~-pent-4-enofuranosyl)-4-ethosy-5-methyl-2-oxopyrimidine, 1-(2,3,5-trideo~y-~-pent-4-enofuranosyl)-4-isoproposy-5-methyl-2-o~opyrimidine, 1-(2,3,5-trideo~y-~-pent-4-enofuranosyl)-5-methyl-2-osopyrimidine, 9-(2,3,5-trideosy-~-pent-4-enofuranosyl)-6-hydroxy-purine, 9-(2,3,5-trideosy-~-pent-4-enofuranosyl)-2,6-di-hydrosypurine.
2',3',5'-trideosyadenosin-3'-ene, 2',3',5'-trideosyuridin-3'-ene, 2',3',5'-trideosycytidin-3'-ene, 2',3',5'-trideosyguanosin-3'-ene, 1-(2,3,5-trideosy-~-pent-3-enofuranosyl)-4-amino-5-methyl-2-osopyrimidine, 1-~2,3,5-trideosy-~-pent-3-enofuranosyl)-g-sthosy-5-methyl-2-osopyrimidine, 1-(2,3,5-tride~sy-~-pent-3-enofuranosyl)-4-isoproposy-5-methyl-2-osopyrimidine, 1-(2,3,5-trideosy-~-pent-3-enofuranosyl)-5-methyl-2-osopyrimidine, 9-(2,3,5-trideosy-~-pent-3-enofuranosyl)-6-hydroxy-purine, and 9-(2,3,5-trideosy-~-pent-3-enofuranosyl)-2,6-di-hydrosypurine.

3'-O-P-AnisoYl-4'-azido-5'-deoxY-5'-iodothYmidine lA. Formula II Where B is Thymine, Z is Iodomethyl ~ ' is O-P-anisoYl and X is H

A solution of 4'-azido-5'-deoxy-5'-iodothymidine 0733M (now 07330~ 26530-FF

ZC~3~08 (11 g, 28 mM), a compound of Formula 4 prepared, for e2ample, as described in Preparation 3A, and p-anisoyl chloride (5.3 g, 31 mM) in pyridine (100 mL) was kept at 21C for 16 hours. Methanol (10 mL) was added and the solution was concentrated by evaporation to a viscous syrup. The syrup was chromatographed on a column of silica-gel (800 g) using 2% methanol in dichloromethane as eluent. Pure 3'-O-p-anisoyl-4'-azido-5'-deoxy-5'-iodo-thymidine ~the title compound) ~13g, 95~) was recovered as a foam which crystallized ~rom ethanol (mp 153-154).

1~. Formula II varYinq B
By following the procedure of part A and substituting for 4'-azido-5'-deosy-5'-iodothymidine the following:
4'-azido-2',5'-dideosy-5'-iodoadenosine, 4'-azido-2',5'-dideoxy-5'-iodoguanosine, 4'-azido-2',5'-dideoxy-5'-iodouridine, 4'-azido-2',5'-dideoxy-5'-iodocytidine, 1-(4-azido-2,5-dideoxy-5-iodo-~-D-erythro-pentofusanosyl)-4-amino-5-methyl-2-oxopyrimidine, 1-~4-azido-2,5-dideosy-5-iodo-~-D-erythro-pentofuranosyl)-4-ethosy-5-methyl-2-oxopyrimidine, 1-(4-azido-2,5-dideoxy-5-iodo-~-D-erythro-pentofuranosyl)-4-isopropoxy-5-methyl-2-oxopyrimidine, 1-(4-azido-2,5-dideoxy-5-iodo-~-D-erythro-pentofuranosyl)-5-methyl-2-oxopyrimidine, - 9-(4-azido-2,5-dideoxy-5-iodo-~-D-erythro-pentofuranosyl)-6-hydrosypurine, and 9-(4-azido-2,5-dideoxy-5-iodo-~-D-erythro-pentofuranosyl)-2,6-dihydroxypurine;

there are obtained the following respective compounds:
~ 6,o3 -di-p-anisoyl-4'-azido-2~,5'-dideoxy-5'-iodoadenosine, 0733M (now 07330) 26530-FF

' :: ' : :~
.

C~3408 N6,N6,o3 -tri-p-anisoyl-4'-azido-2',5'-dideoxy-5'-iodoadenosine, N2,o3 -di-p-anisoyl-4'-azido-2',5'-dideoxy-5'-iodoguanosine, N2,N2,o3 -tri-p-anisoyl-4'-azido-2',5'-dideoxy-5'-iodoguanosine, 0 -p-anisoyl-4'-azido-2',5'-dideoxy-5'-iodouridine, 223-225C dec., N4,03-di-p-anisoyl-4'-azido-2',5'-dideo~y-5'-iodocytidine, N4,N4,03-tri-p-anisoyl-4'-azido-2',5'-dideoxy-5'-iodocytidine, N4-p-anisoyl-1-(03-p-anisoyl-4-azido-2,5-dideoxy-5-iodo-~-D-erythro-pentofuranosyl)-4-amino-5-methyl-2-oso-pyrimidine, N4,N4-di-p-anisoyl-1-~03-p-anisoyl-4-azido-2,5-dideosy-5-iodo-~-D-erythro-pentofuranosyl)-4-am~no-5-methyl-2-oso-pyrimidine, 1-(0 -p-anisoyl-4-azido-2,5-dideoxy-5-iodo-~-D-erythro-pentofuranosyl)-4-etho~y-5-methyl-2-oxo-pyrimidine, 1-(03-p-anisoyl-4-azido-2,5-dideoxy-5-iodo-~-D-erythro-pentofuranosyl)4-isopropoxy-5-methyl-2-oso-pyrimidine, 06-p-anisoyl-9-(03-p-anisoyl-4-azido-2,5-dideoxy-5 -iodo-~-D-erythro-pentofuranosyl)6-hydrosypurine, and 02,06-di-p-anisoyl-9-(03-p-anisoyl-4-azido-2,5-d ideo~y-5-iodo-~-D-erythro-pentofuranosyl)2,6-di-hydro~ypurine, and 1-(03-p-anisoyl-4-azido-2,5-dideoxy-5-iodo-~-D-erythro-pentofuranosyl)5-iodo-5-methyl-2-oxo-pyrimidine.

lC. Formula II Where Y' is 0-benzoYl ~ y following the procedure of part A and substituting for p-anisoyl chloride an equivalent amount of benzoyl 0733M (now 07330) 26530-FF

-- 2~ 408 chloride, there is obtained 4'-azido-3'-O-benzoyl-5'-deosy-5'-iodothymidine, ~5,N6,o3 -tribenzoyl-4'-azido-2',5'-deosy-5'-iosoadenosine, and the other appropriate compounds named in part A.

4'-Azidothvmidine 2A. Formula I Where B is Thymine, n is Zero, Y~ is Hvdroxvl. and X is HYdroaen A solution of 3-chloroperosybenzoic acid (85% grade, 21.0 g, 103mM) in dichloromethane saturated with H20 tl50 mL) was added to a stirred solution of 3 '-0-p-ani80~1-4 ' -azido-5'-deo~y-5'-iodothymidine (12.0 g, 22.8mM), prepared, e.g., as described in E~ample 1, in dichloromethane saturated with H2O
(150 mL). .~fter 3 hours at 21~, the reaction mixture was diluted with dichloromethane (200 mL) and n-butanol (50 mL), and then estracted successively with saturated agueous sodium bisulfite (100 mL), saturated aqueous sodium bicarbonate (100 mL), and H20 (100 mL). The organic phase was then concentrated in vacuo to a viscous syrup. A solution of the syrup in methanol (250 mL) and concentrated ammonium hydroside (250 mL) was kept at 21C
for 16 hours and then the solvent was removed by evaporation. The residue was chromatographed on a column of silica-gel (500 g) and eluted with 8% methanol in dichloromethane. Pure product was recovered and crystallized from ethanol to give the title compound, 4'-azidothymidine (1.46 q, 23%) (mp 175-176) .
0733M (now 0733~) 26530-FF

20~ 08 2B. Formula I VarYina B
By following the procedure of part A and substituting for 3'-Q-p-anisoyl-4'-azido-5'-deoxy-5'-iodothymidine the following:
N6,o3 -di-p-anisoyl-4'-azido-2',5'-dideoxy-5'-iodoadenosine, N6,N6,o3 -tri-p-anisoyl-4~-azido-2',5'-dideo~y-5'-iodoadenosine, N2,o3 -di-p-anisoyl-4'-azido-2',5'-dideo~y-5'-iodoguanosine, N2,N2,o3 -tri-p-anisoyl-4'-azido-2',5'-dideoxy-S'-iodoguanosine, 0 -p-anisoyl-4'-azido-2',5'-dideosy-5'-iodouridine, N4,03~di-p-anisoyl-4'-azido-2',5'-dideoxy-5'-iodocytidine;
N4,N4,03-tri-p-anisoyl-4'-azido-2',5'-dideoxy-5'-iodocytidine, N4-p-anisoyl-1-(03-p-anisoyl-4-azido-2,5-dideoxy-5-iodo-~-D-erythro-pentofuranosyl)-4-amino-5-methyl-2-oxo-pyrimidine, N4,N4-di-p-anisoyl-1-~03-p-anisoyl-4-azido-2,5-dideoxy-5-iodo-~-D-erythro-pentofuranosyl)-4-amino-5-methyl-2-oxo-pyrimidine, 1-(03-p-anisoyl-4-azido-2,5-dideoxy-5-iodo-~-D-erythro-pentofuranosyl)-4-ethoxy-5-methyl-2-oxo-pyrimidine, 1-(03-p-anisoyl-4-azido-2,5-dideoxy-5-iodo-~-D-erythro-pentofuranosyl)4-isopropoxy-5-methyl-2-oxo-pyrimidine, 06-p-anisoyl-9-(03-p-anisoyl-4-azido-2,5-dideoxy-5 -iodo-~-D-erythro-pentofuranosyl)6-hydroxypurine, and 02,06-di-p-anisoyl-9-(03-p-anisoyl-4-azido-2,5-d ideoxy-5-iodo-~-D-erythro-pentofuranosyl)2,6-di-hydroxypurine, and 0733M (now 07330~ 26530-FF

2Cg)3~08 1-(03-p-anisoyl-4-azido-2,5-dideoxy-5-i.odo-~-D-erythro-pentofuranosyl)5-iodo-5-methyl-2-oso-pyrimidine.

there are obtained the following respective compounds:
4'-azido-2'-deosyadenosine (mp 92-105C), 4'-azido-2'-deosyguanosine (mp 2500C, w/decomposition), 4'-azido-2'-deosyuridine (mp 272-273C), 4'-azido-2'-deosycytidine (mp 84-85C), 1-(4-azido-2-deoxy-~-D-erythro-pentofuranosyl)-4-amino-5-methyl-2-osopyrimidine, 1-(4-azido-2-deoxy-~-D-erythro-pentofuranosyl)-4-ethosy-5-methyl-2-oxopyrimidine, 1-(4-azido-2-deosy-~-D-erythro-pentofuranosyl)-4-isoproposy-5-methyl-2-osopyrimidine, 1-(4-azido-2-deoxy-~-D-erythro-pentofuranosyl)-5-methyl-2-osopyrimidine, 9-(4-azido-2-deoxy-~-D-erythro-pentofuranosyl)-6-hydrosypurine, and 9-(4-azido-2-deoxy-~-D-erythro-pentofuranosyl)-2,6-dihydrosypurine~

2C. Formula I Made From Formula II Where Y' is 0-benzovl By following the pro~edure of part A and substituting ~or 3'-0-p-anisoyl-4'-azido-5'-deoxy-5'-iodothymidine an eguivalent amount of 4'-azido-3'-0-benzoyl-5'-deoxy-5'-iodothymidine, there is obtained 4'-azidothymidine.

0733M (now 07330) 26530-FF

ZC~3408 EXA~L3~: 3 1-~3-Deo~Y-4-methox~-a-L-alYcero-DentofuranosYl)thYmidine 3A. Formula III Where R3 is CH3;
B is ThYmine: and X and Y' are H
3',5'-Dideosythymidin-4'-ene (30 mg, 0.144 mM) was disæol~ed in 1.0 mL of methanol and kept under nitrogen.
Solid potassium bicarbonate (36 mg, 0.36 mM) was added and the misture was cooled in an ice/water bath.
3-Chloroperosybenzoic acid (0.214 mM) was added in one lot and the misture stirred for 3 min. before the reaction was quenched by the addition of 2.0 mL of a 1:1 misture of saturated sodium bicarbonate and 10% (w/v) sodium thiosulfate in water. The resulting misture was estracted twice with 50 mL of ethyl acetate:chloroform (1:3 v/v). The estracts were washed with 2.0 mL of brine, combined, dried over magnesium sulfate and concentrated in vacuo. The residue (31 mg) was chromatographed on two silica gel plates giving 1-(3-deosy-4-methos~ -glycero-pentofuranosyl)thymidine (8 mg, 31 ~M, 22~) as an oil.

3B. Formula III Var~ina B
~ y following the procedure of part A and substituting 3',5'-dideosythymidin-4'-ene with the following:
2',3',5'-trideoxyadenosin-4'-ene, 2',3',5'-trideosyuridin-4'-ene, 2',3',5'-trideosycytidin-4'-ene, 2~,3',5'-trideosyguanosin-4'-ene, 1-(2,3,5-trideosy-~-pent-4-enofuranosyl)-4-amino-5-methyl-2-osopyrimidine, 1-(2,3,5-trideoxy-~-pent-4-enofuranosyl)-4-ethoxy-5-methyl-2-oxopyrimidine, 0733M (now 07330) 26530-FF

1-(2,3,5-trideosy-~-pent-4-enofuranosyl)-4-isoproposy-5-methyl-2-osopyrimidine, 1-~2,3,5-trideosy-~-pent-4-enofuranosyl)-5-methyl-2-osopyrimidine, 9-(2,3,5-trideo~y-~-pent-4-enofuranosyl)-6-hydrosypurine, and 9-(2,3,5-trideosy-~-pent-4-enofuranosyl)-2,6-dihydrosypurine;

there are obtained the following respective compounds:
9-(2,3-dideosy-4-methoxy-a-L-glycero-pentofuranosyl)adenine, 1-(2,3-dideosy-4-methosy-a-L-glycero-pentofuranosyl)uracil, 1-(2,3-dideosy-4-methosy-a-L-glycero-pentofuranosyl)cytosine, 9-(2,3-dideosy-4-methosy-a-L-glycero-pentofuranosyl)guanine, (2,3-dideosy-4-methosy-a-L-glycero-pentofuranosyl)4-amino-5-methyl-2-oxopyrimidine, 1-(2,3-dideosy-4-methosy-a-L-glycero-pentofuranosyl)-4-ethosy-5-methyl-2-osopyrimidine, 1-(2,3-dideosy-4-methosy-a-L-glycero-pentofuranosyl)-4-isoproposy-5-methyl-2-oxo-pyrimidine, 1-(2,3-dideosy-4-methosy-a-L-glycero-pentofuranosyl)-5-methyl-2-osopyrimidine, 9-(2,3-dideosy-4-methoxy-a-L-glycero-pentofuranosyl)-6-hydrosypurine, and 9-(2,3-dideosy-4-methosy-a-L-glycero-pentofuranosyl)-2,6-dihydrosypurine.

:
. .
':

0733M (now 07330) 26530-FF
.~ .

2C~);3408 4'-Azi~o-3'-deo~YthYmidine 4A. Formula I Where B is Thymine; X and Y' are ~;
and n is Zero 1-(2,3-dideosy-4-methoxy-~-L-glycero-pento-furanosyl)thymine (19 mg, 74 ~M) was dissolved in 2 mL
of dimethylformamide under nitrogen and 31 mg (0.45 mM) of imidazole and 31 mg ~0.21 mM) of t-butyldimethylsilyl chloride were added. The misture was stirred at room temperature for 3 hours before the reaction was quenched by the addition of 2 drops of methanol. The mixture was poured into 30 mL of saturated sodium bicarbonate and extracted twice with 50 mL of ethyl acetate. The estracts were washed with brine, dried over magnesium sulfate and concentrated in vacuo. The oily residue (40 mg) was chromatographed on a silica gel plate using chloroorm containing 4% of methanol as the eluent.
Isolation of the product band gave a silyl ether, 1-~2,3-dideosy-5-O-(l,l-dimethylethyl)-dimethyl-silyl-4-methosy-a-L-glycero-pentofuranosyl]thymine (24 mg, 65 ~M, 89~) as an oil.
The nest step was carried out in two batches; 16 mg and 7 mg of 8ilyl ether respectively. The two were combined for workup and purification. The procedure for the larger batch is described below.
The silyl ether (16 mg, 43~M) was dissolved in 1.5 mL of methylene chloride and azidotrimethylsilane (45~L) and trimethylsilyl trifluoromethanesulfonate (6~L) were added.
The mixture was stirred for 72 hours at room temperature and combined with the reaction mixture from the 7 mg batch. Dilution with 50 mL of ethyl acetate:
chloroform (1:3 v/v) and washing with 5 mL of saturated 0733M (now ~7330) 26530-FF

~, , ~3408 sodium bicarbonate followed by 5 mL of brine and drying over magnesium sulfate and subsequent evaporation gave 16 mg of an oil. Chromatography of the oil on silica gel plates followed by crystallization from ethyl acetate:
; hesane gave 4~-azido-3~-deosythymidine ~5 mg, mp 55-60C
decomp.) and 1-(4-azido-2,3-dideosy--L-glycero-pento-furanosyl)thymine (3 mg, mp 129-131C).

4B. Formula I-B varYina B
By following the procedure of part A and substituting 1-(2,3-dideosy-4-methosy-a-L-glycero-pentofuranosyl)-thymine with the following:
9-(2,3-dideosy-4-methosy--L-glycero-pentofuranosyl)adenine, 1-(2,3-dideosy-4-methosy--L-glycero-pentofuranosyl)uracil, 1-(2,3-dideosy-4-methosy--L-glycero-pentofuranosyl)cytosine, 9-(2,3-dideosy-4-methosy--L-glycero-pentofurano~yl)guanine, 1-~2,3-dideosy-4-methosy--L-glycero-pentofuranosyl)4-amino-5-methyl-2-osopyrimidine, 1-(2,3-dideosy-4-methosy--L-glycero-pentofuranosyl)-4-ethosy-5-methyl-2-osopyrimidine, :1-(2,3-dideosy-4-methosy-a-L-glycero-pentofuranosyl)-4-i 80p roposy-5-methyl-2-oso-pyrimidine, 1-(2,3-dideosy-4-methosy--L-glycero-pentofuranosyl)-5-methyl-2-osopyrimidine, :9-(2,3-dideosy-4-methosy--L-glycero--pentofuranosyl)-6-hydrosypurine, and ;9-(2,3-dideosy-4-methosy--L-glycero-pentofuranosyl)-2,6-dihydroxypurine;

there are obtained the following respective compounds:
4'-azido-2',3'-dideoxyadenosine, 0733M (now 07330) 26530~FF

2C~3408 4'-azido-2',3'-dideo~yuridine, 4~-azido-2',3'-dideosycytidine, 4'-azido-2',3'-dideosyguanosine, 1-(4-azido-2,3-dideosy-~-D-glycero-pentofuranosyl)-4-amino-5-methyl-2-osopyrimidine, 1-(4-azido-2,3-dideo~y-~-D-glycero-pentofuranosyl)-4-ethosy-5-methyl-2-osopyrimidine, l-t4-azido-2,3-dideosy-~-D-glycero-pentofuranosyl)-4-isoproposy-5-methyl-2-osopyrimidine, 1-(4-azido-2,3-dideo~y-~-D-glycero-pentofuranosyl)-5-methyl-2-osopyrimidine, 9-(4-azido-2,3-dideosy-~-D-glycero-pentofuranosyl)-6-hydrosypurine, and 9-(4-azido-2,3-dideosy-~-D-glycero-pentofuranosyl)-2,6-dihydrosypurine.

4'-Azidothymidine 5'-Mono~hosDhate Disodium Salt To a suspension of 4'-azidothymidine (O.20 g, 0.71 mM) in ethyl acetate (12 mL) cooled to 0C was added pyrophosphoryl chloride (0.5 mL, 3.7 mM). After stirring for 4 hours at 0C, the solution was neutralized (pH 6 to 7) by the addition of saturated aqueous sodium bicarbonate. The organic phase was discarded, and the agueous phase was stirred for 20 minutes with activated, 14-60 mesh, charcoal. The charcoal mixture was filtered and the charcoal rinsed with water ~200 mL). All filtrates and rinsings were discarded. The charcoal was nest washed with 50% aqueous ethanol containing 5%
ammonium hydroside (250 mL). These washings were concentrated in vacuo to about 5 mL and applied onto a 3 0733M (now 07330) 26530-FF

:., , '. ' '- ,' ~

: ~ - .. . .
~. ' . : .

` ZC~3408 x 23 cm column of DEAE Sephadex (carbonate form). The column was eluted with a linear gradient consisting of lL
of water and 1 L of 0.5 M triethylammonium bicarbonate (pH 7). Pure product waF collected and the solvent removed by evaporation. The residue was co-evaporated repeatedly with water, then dissolved in methanol (0.5 mL). The addition of 0.45 M sodium perchlorate in acetone (2 mL) to this solution caused the title compound to precipitate out. Filtering off the solid and drying under vacuum left 55 mg of 4'-azidothymidine 5'-monophosphate disodium salt (mp 185C, decomposition).

.

4~-Azidothymidine 5'-Diphosphate Trisodium Salt and 4'-Azidothvmine 5'-TriPhosDhate Tetrasodium Salt /
A solution of 4'-azidothymidine 5'-monophosphate disodium salt (55 mg, 0.14 mM) in H2O (5 mL) is applied onto a short column of Dowex 50 (H+) resin (8 mL) and eluted with H2O. After concentrating the eluent to 5 mL, tributylamine (32mL) and pyridine (5 mL) was added and the mixture evaporated in vacuo. The residue was co-evaporated with pyridine then with dimethylformamide. To a solution of the residue in dimethylformamide (1 mL) was added a solution of carbonyldiimidazole (0.11 g, 0.66 mM) in dimethyl-formamide (1.5 mL). After 20 hours at room temperature, methanol (0.04 mL, 1.0 mM) was added. Thirty minutes later a solution of tributylammonium pyrophosphate (0.675 mM) in dimethylformamide (7 mL) was added and the mixture was vigorously stirred for 20 hours at room temperature. After filtration, the solution was diluted with an equal volume of methanol and evaporated to a 07330 (now 1548F) 26530-FF

- ZC~3~08 syrup. The syrup was dissolved in water (lO mL) and applied onto a 3 x 23cm column of DEAE Sephadex (carbonate form) which was eluted with a linear gradient consisting of 2L of water and 2L of 0.5M triethylammonium bicarbonate (pH 7). The 4'-azidothymidine 5'-diphosphate was eluted pure followed by the 4'-azidothymidine 5-'triphosphate.
After evaporation of solvent and repeated co-evaporation with water, each product was dissolved in a small amount of methanol. The addition of a 0.45 M solution of sodium perchlorate in acetone then precipitated the sodium salts.
Filtering off the precipitates and drying in vacuo gave 4'-azidothymidine 5'-diphosphate trisodium salt (12 mg~
and 4'-azidothymidine 5'-triphosphate tetrasodium salt (54 mg, mp 204C, decomposition).

4 ' -AZidOthYmidine 3 ', S ' -CYC1iC PhosPhate Sodium Salt 4'-Azidothymidine 5'-monophosphate disodium salt, prepared, e.g., as described in E~ample 5, is passed through a column of Dowex 50-X8 cation-exchange resin (H+ form) with water as eluent. Upon evaporation of the water, the residue is dissolved in a mixture of pyridine and water containing N,N'-di-cyclohexyl-4-morpholinecarboxamidine, and is then evaporated in vacuo. The residue is co-evaporated three times with pyridine. A solution of the resulting syrup in pyridine is added dropwise over 1 hour to a refluxing solution of dicyclohexylcarbodiimidate in pyridine. When the addition is complete, the mixture is heated at reflux for an additional 2.5 hours, followed by removal of the solvent by evaporation in vacuo. A solution of the residue in water is extracted with 10% butanol in 07330 (now 1548F) 26530-FF

20~3~08 dichloromethane, and then with diethyl ether. The aqueous solution is concentrated by evaporation and applied onto a 3x23 cm column of DEAE Sephadex (carbonate form) which is eluted with a linear gradient consisting of 1 L of water and 1 L of 0.5 M triethylammonium bicarbonate ~pH 7). Eluent containing the pure product is evaporated in vacuo, and the residue co-evaporated repeatedly with water. To a concentrated solution of the residue in methanol is added a 0.45 M solution of sodium perchlorate in acetone. The precipitated product is removed and dried to give the title compound 4'-azido-thymidine 3',5'-cyclic phosphate sodium salt, mp 155-157C.
In a similar manner, the other compounds of Formula I where n is 1 can be converted to the cyclic phosphate form. By substituting calcium chloride for sodium perchlorate, the 4'-azidothymidine 3',5'-cyclic phosphate calcium salt is obtained.

Sodium Salt of 4'-Azidothvmidine 4'-Azidothymidine is dissolved in water. One molar equivalent of sodium hydroxide in water is added and the solution is stirred for 1 hour. The solution is then lyophilized to isolate the sodium salt of 4'-azidothymidine.
In a similar manner, all compounds of Formula I
where n is zero may be converted to the base addition salts by treatment with the appropriate base, for example, sodium hydroxide, potassium hydroxide, lithium hydroxide, and the like.

07330 (now 1548F) 26530-FF

2~ 4~)8 9A. 3'.5'-Di-adamantoYl Ester of 4~-AzidothYmidine 4'-Azidothymidine (280 mg) and 10 mg of 4-dimethyl-aminopyridine in a solution of 7 ml of pyridine is added 2.1 equivalents of adamantanecarboxylic acid chloride as a solution in 3 ml of methylene chloride. The solution is magnetically stirred for 18 hours at 21C and 1 ml of methanol is then added. The solution is concentrated and the residue is purified by chromatography to give the title compound, 4'-azidothymidine 3',5'-diadamantoate.

9B. 3',5'-di-O-acetYl ester of 4'-Azidothvmidine To a solution of 4'-azidothymidine (1.00 g, 3.53 mM) in pyridine (10 ml) is added acetic anhydride (2.0 ml, 21 mM). After 2 hours at 20C, the solvent is removed by evaporation. The residue is purified by chromatography on silica gel using a gradient elution of 2% to 4%
methanol in dichloromethane. Pure 4'-azido-3',5'-di-O-acetylthymidine is isolated as 0.92 g (71% yield) of white foam. lH NMR (CDC13) gave 8.94 (lH, broad s, ~H), 7.17 (lH, d, H-6), 6.42 (lH, dd, H-l~), 4.41 (2H, s, H-5'), 2.55 (2H, m, H-2'), 2.17 (3H, s, OAc), 2.16 (3H, s, OAc), 1.96 (3H, d, CH3).

9C. 3' 5'-di-O-acetvl ester of 4'-Azido-2'-deoxv-Uridine A solution of 4'azido-2'-deoxy-uridine (269 mg, 1.00 mM), acetic anhydride (1.0 ml, 10.6 mM), and pyridine (6 ml) is kept at room temperature for 4 hours and therl evaporated in vacuo to a syrup. The syrup is pur;fied by thin layer chromatography on silica gel using 5% methanol in dichloromethane as eluent. The product is 07330 (now 1548F) 26530-FF

20C)3408 crystallized from ethanol to give 334 mg (95% yield) of 4'-azido-2'-deoxy-3',5'-di-O-acetyluridine, mp 136-137C.

9D. 5'-O-benzoYl ester of 4'azidothYmidine A solution of 4'azidothymidine (140 mg, 0.5 mM~, benzoyl chloride (70 ~1, 0.6 mM), and pyridine (1.0 ml) is kept at 20C for 3 hours. Following the addition of 0.1 ml of methanol, the solvent is removed by evaporation. The residue is purified by preparative thin layer chromatography on silica gel, eluting with 5%
methanol in dichloromethane. The product (4'-azido-5'-O-benzoylthymidine) is isolated as 0.10 g (53% yield) of white foam. lH NMR ~CDC13) gave 11.37 (lH, broad s, NH), 8.05-7.52 (5H, m, Ph), 7.44 (lH, d, H-6), 6.36 (lH, dd, H-l'), 5.98 (lH, broad s, OH), 4.73 (lH, m, H-3~), 4.65 (lH, d, H-5'), 4.55 (lH, d, H-5'j, 2.40 (2H, m, H-2'), 1.70 (3H, d, CH3)-10A. 1-(4'azido-2-deoxy-3,5-di-O-acetyl-~-D-erythro-pentofuranosyl)-5-methyl-4-(1,2,4-triazol-1-yl)pyrimidin-2(lH)-one.

To a suspension of triazole (693 mg, 10.0 mM) in acetonitrile (6.0 ml) is added phosphoryl chloride (226 pl, 2.43 mM). The mixture is cooled to 0C and then triethylamine (1.26 ml, 9.3 mM) is slowly added. Ater 10 minutes at 0C, a solution of 4'-azido-3',5'-di-O-acetylthymidine (460 mg, 1.26 mM) in acetonitrile (4.0 ml) is added and the mixture is brought to room temperature. After stirring for 2 hours, the solids are removed by filtration and the filtrate is concentrated by evaporation. The solution is purified by preparative 07330 (now 1548F) 26530-FF

- ZC~3~10 thin layer chromatography on silica gel, eluting with 5%
methanol in dichloromethane. The isolated product is crystallized from methanol affording 474 mg (90% yield) of the title compound, mp 109-111C.

10B. 1-(4-azido-2-deoxy-~-D-erythro-pentofuranosyl)-4-ethoxY-5-methYlPvrimidin-2-(lH~-one.

A solution of 1-(4-azido-2-deoxy-3,5-di-O-acetyl-~D-erythro-pentofuranosyl)-5-methyl-4-(1,2,4-triazol-1-yl)pyrimidin-2(1H)-one (42 mg, 0.1 mM) in a lN solution of sodium ethoxide in ethanol (1.0 ml) is kept at room temperature for 2 hours. The solution is applied directly onto a preparative thin layer silica gel chroma~ography plate and eluted with dichloromethane/methanol 9:1. The pure title compound is isolated as 26 mg (84% yield) of white foam. lH NMR
(DMSO-d6) gave 7.93 (lH, d, H-6), 6.30 (lH, dd, H-l'), 5.74 (lH, d, )H), 5.64 (lH, t, OH), 4.45 (lH, m, H-3'), 4.32 (2H, q, )CH2CH3), 3.66 (2H, m, H-5'), 2.31 (2H, m, H-2'), 1.89 (3H, d, CH3), 1.31 (3H, t, OCH2CH3).

10C. 1-(4-Azido-2-deoxy-~-D-erythro-pentofuranosyl)-4-isoProPoxv-5-methYlPyrimidin-2~lH~-one.

A solution of 1-(4-azido-2-deoxy-3,5-di-O-acetyl-D-erythro-pentofuranosyl)-5-methyl-4-(1,2,4-triazol-l-yl)pyrimidin-2(1H)-one (168 mg, 0.40 mM) in a lN
solution of potassium isopropoxide in isopropanol (4.0 ml) is kept at room temperature for 1.5 hours. The solution is neutralized with Dowex 50 (H+) resin and filtered. The filtrate is concentrated in vacuo to an oil which is purified by preparative thin layer chromatography using 10% methanol in dichloromethane as 07330 (now 1548F) 26530-FF

ZQ~34n~

eluent. The pure title compound is obtained as 105 mg (80% yield) of white foam. lH NMR (DMSO-d6) gave 7.91 (lH, d, H-6), 6.30 (lH, dd, H-l'), 5.74 (lH, d, OH), 5.63 (lH, t, OH), 5.26 (lH, m, OCH(CH3)2), 4.45 (lH, m, H-3'), 3.66 (2H, m, H-5'), 2.31 (2H, m, H-2'), 1.86 (3H, d, CH3), 1.30 (3H, s, CH3), 1.28 (3H, s, CH3).

10D. 1-(4-Azido-2-deoxy-~-D-erythro-pentofuranosyl)-5-methYl~Yrimidin-2-(1H~-one.

A solution of 1-(4-azido-2-deoxy-3,5- di-O-acetyl-~-D-erythro-pentofuranosyl)-5- methyl-4-(1,2,4-triazol-l-yl)pyrimidin-2(1H)-one (335 mg, 0.80 mM), hydrazine hydrate (160 ml, 3.2 mM), and acetonitrile (6.0 ml) is kept at 20C for 1 hour and then evaporated in vacuo to a syrup. The syrup is chromatographed on thin layer silica gel, eluting with dichloromethane/methanol 85:15 containing 1% concentrated ammonium hydroxide. The major product, isolated as a white powder, is heated at reflux in ethanol (15 ml) in the presence of silver (I) oxide (300 mg). After 2 hours, the filtered reaction mixture is evaporated in vacuo to a syrup. The syrup is purified by preparative thin layer chromatography on silica gel, eluting with dichloromethane/methanol 85:15 containing 1%
concentrated ammonium hydroxide. The isolated product is crystallized from ethyl acetate affording 88 mg (41%
yield) of the title compound, mp 143-144C.

10E. 4'-Azido-2'-deoxY-5-methYlcYtidine A solution of 1-(4-azido-2-deoxy-3,5-di-O-acetyl-D-erythro-pentofuranosyl)-5-methyl-4-(1,2,4-triazol-l-yl~pyrimidin-2(lH)-one (21 mg, 50 ~ ), dioxane (0.20 ml), and concentrated ammonium hydroxide (0.20 ml) is kept at room temperature for 2.5 hours. The solution is 07330 (now 1548F) 26530-FF

` ZC9~408 applied directly onto a preparative thin layer silica gel-chromatography plate and eluted with dichloromethane/methanol 85:15 containing 1~ concentrated ammonium hydroxide. The product is isolated and crystallized from ethanol/ethyl acetate to afford 10 mg (71% yield) of the title compound, mp 132-134C.
lOE. 4'-Azido-2'-deoxYcYtidine To a suspension of triazole (480 mg, 6.96 mM) in acetonitrile (4.0 ml) is added phosphoryl chloride (150 pl, 1.61 mM). The mixture is cooled to 0C and then triethylamine (0.91 ml, 6.46 mM) is slowly added. After 10 minutes at 0C, a solution of 4'-azido-2'-deoxy-3',5'-di-O-acetyluridine (300 mg, 0.85 mM) in acetonitrile (2.5 ml) is added and the mixture is brought to room temperature. After stirring for 2 hours, the solids are removed by filtration and the filtrate is concentrated by evaporation. The residue is chromatographed on preparative silica gel plates using 4% methanol in dichloromethane as eluent. The major product is isolated as 240 mg of white foam. A solution of the foam in dioxane (4.0 ml) and concentrated ammonium hydroxide (8.0 ml) is kept at room temperature for 16 hours. The solution is concentrated in vacuo and purified by preparative thin layer silica gel chromatography using ethyl acetate/propanol/water 5.5:1.5:3 (organic phase) as eluent. The product is isolated as a foam which is triturated with ethyl acetate to provide 160 mg (70%
yield) of 4'-azido-2'-deoxycytidine as a white powder, mp 84-85C.

Similarly, by substituting for adamantanecarboxylic acid chloride the appropriate number of molar equivalents of the appropriate acid chloride, there is obtained 07330 (now 1548F) 26530-FF

,, . ~ , .
. .

-` ZC~08 4'-azidothymidine 3',5'-dipalmitoate, 4'azidothymidine 3',5'-dibenzoate, 1-(4-azido-2,3-dideoxy-a-L-glycero-pentofuranosyl)-thymine 5' acetate, or 1-(4-azido-2,3-dideoxy-a-L-glycero-pento-furanosyl)-thymine 5' anisoate.

.

4'-Azido-3' 5'-di-0-~3-DvridinYlcarbonyl)thYmidine 4'-Azidothymidine (1.05 g, 3.71 mM) is dissolved in dry pyridine (35 ml) under nitrogen. Nicotinoyl chloride hydrochloride (1.45 g, 8.15 mM) followed by 4-dimethylaminopyridine (0.1 g, O.82 mM) and triethylamine (1.5 ml, 8.25 mM) is added under stirring.
The mi~ture is stirred at room temperature for 5 hours and the reaction is quenched by the addition of methanol (0.5 ml). The mixture is poured into an aqueous, saturated sodium bicarbonate solution and extracted with ethyl acetate. The organic phase is washed with brine and dried over magnesium sulfate. Following the evaporation of the solvent, the oily residue is chromatographed on a silica gel column using ethyl acetate/hexane mixture as the eluting solvent. The fractions containing the product are combined and evaporated. The residue is crystallized from an ethyl acetate/hexane mixture to give 4'-azido-3',5'-di-O-(3-pyridinylcarbonyl)thymidine, mp 204-206C.

07330 (now 1548F) 26530-FF

2C~ 408 4'-Azido-5'-0-(1 4-dihYdro-l-methYl-3-~YridinYlcarbonYl)thvmidine 12A. 4'-Azido'5'-0-(3-~vridinYlcarbonvlL~idine 4'-Azidothymidine (1.05 g, 3.7 mM) is dissolved in dry pyridine (35 mL) and nicotinoyl chloride hydrochloride (0.8 g, 4.4 mM) is added followed by triethylamine (0.62 ml, 4.4 mM). The mixture is maintained at room temperature for 3 hours. The pyridine is removed under vacuo and the remaining residue is purified by chromatography. The appropriate fractions are combined to give, after evaporation of the eluent, 4'-azido-5'-0-(3-pyridinylcarbonyl)thymidine.

12B. 4'-Azido-5'-0-(1-methyl-3-pyridinium-carbonYl~thYmidine 4'-Azido-5'-0-(1-methyl-3-pyridiniumcarbonyl)-thymidine (69 mg, 0.18 mM) is dissolved in acetone (3 ml) and iodomethane (0.1 ml, 1.6 mM) is added. The mixture is refluxed for 4.5 hours and iodomethane (0.05 ml, 0.8 mM) is added. After an additional 2 hours oE heating, the mixture is allowed to cool to room temperature. A
residue is formed and hexane (3 ml) is added to precipitate more product. After removal of the mother liquor, 4'-azido~5'-0-(1-methyl-3-pyridiniumcarbonyl)-thymidine is isolated as a yellow solid, mp 130-134C.

07330 ~now 1548F) 26530-FF

.

2C~34~8 12C. 4'-Azido-5'-0-(1,4-dihydlo-1-methyl-3-pyridinyl-carbonvl)thvmidine.

An aqueous solution containing sodium hydrosulfite S0.2 M) and sodium bicarbonate (0.2 M) is deaerated by bubbling a stream of argon gas through the solution for 2 hours at room temperature. This solution (2.5 ml) is added under argon to 4'-azido-5'-O-(l-methyl-3-pyridiniumcarbonyl)thymidine (45 mg, 0.085 mM) at room temperature. The resulting mixture is stirred at room temperature for 30 minutes. The supernatant is rçmoved from the light yellow precipitate, which is dried under vacuo to give 4'-azido-5'-0-(1,4-dihydro-1-methyl-3'-pyridinylcarbonyl)thymidine, mp 115-127C.

4'-Azido-3' 5'~di-O-iso~roPylthYmidine 4'-Azido-N benzoylthymidine (1 mM) prepared according to the method of M. Sekine et al., SYnthesis 1119 ~1987), is dissolved in dimethylsulfoxide (10 mL) and sodium hydride (2.1 equivalents) is added, and the mixture is stirred at room temperature for 30 minutes.
to this mixture, 2-iodopropane (10 equivalents) is added and the mixture is left at 60C until most of the starting material has disappeared, as monitored by thin layer chromatography. The reaction mixture is partitioned between brine and ethyl acetate. The organic phase is dried and evaporated to dryness. The residue is dissolved in saturated methanolic ammonia and left for 1 hour at room temperature. The solvent is evaporated to 07330 (now 1548F) 26530-FF

;

-- 2(~ )8 dryness and the residue is purified by column chromatography to give the title compound, 4'-azido-3',5'-di-O-isopropylthymidine.

This example illustrates the preparation of a representative pharmaceutical formulation for oral administration containing an active compound of Formula I, e.g., 4'-azidothymidine.
Quantity per Inaredients tablet. mqs.
Active compound 200 lactose, spray-dried 148 magnesium stearate 2 The above ingredients are mixed and introduced into a hard-shell gelatin capsule.
Other compounds of Formula I, such as those prepared in accordance with Examples 2-13, can be used as the active compound in the preparation of the orally administrable formulations of this example.

EXAMPI,E 15 This example illustrates the preparation of a representative pharmaceutical formulation containing an active compound of Formula I, e.g., 4'-azidothymidine.
An suspension for oral administration is prepared 07330 (now 1548F) 26530-FF

.

2C~3~Q8 having the following composition:
Inaredients OuantitY
Active compound 1.0 g fumaric acid 0.5 g sodium chloride . 2.0 g methyl paraben . 0.1 g granulated sugar 25.5 g sorbitol (70% solution) 12.85 g Veegum K (Vanderbilt Co.) 1.0 g flavoring 0.035 mL
colorings 0.5 mg distilled water q.s. to 100 mL

Other compounds of Formula I, such as those prepared in accordance with Examples 2-13, can be used as the active compound in the preparation of the orally administrable formulations of this example.

This example illustrates the preparation of a representative pharmaceutical formulation containing an active compound of Formula I, e.g., 4'-azidothymidine.
An injectable preparation is prepared having the following composition:
Inaredients Active compound 0.2 g water (distilled, sterile) q.s. to 20.0 mL

Other compounds of Formula I, such as those prepared in accordance with Examples 2-13, can be used as the active compound in the preparation of the injectable formulations of this example.
Compounds of Formula I having low solubility in 07330 (now 1548F) 26530-FF

2C~ 8 water can be formulated for intravenous injection in liposomes.

This example illustrates the preparation of a representative pharmaceutical formulation for topical application containing an active compound of Formula I, e.g.~ 4'-azidothymidine.
Inaredients ~rams Active compound 0.2-10 Span 60 2.0 Tween 60 2.0 Mineral oil 5.0 Petrolatum 10.0 Methyl paraben 0.15 Propyl paraben 0.05 BHA (butylated hydroxy anisole) 0.01 Water q.s. to 100 All of the above ingredients, except water, are combined and heated to 60C with stirring. A suf~icient quantity of water at 60C is then added with vigorous stirring to emulsify the ingredients, and water then added, q.s. to 100 g.
Other compounds of Formula I, such as those prepared in accordance with Examples 2-13, can be used as the active compound in the preparation of the topical formulations of this example.

07330 (now 1548F) 26530-FF

.

., ' . ,.. . ' This example illustrates the preparation of a representative pharmaceutical formulation containing an active compound of Formula I, e~g., 4'-azidothymidine.
A suppository totalling 2.5 grams is prepared having the following composition:
Active compound 500 mg witepsol H-15* balance (*triglycerides of saturated vegetable fatty acid; a product of Riches-Nelson, Inc., New York, N.Y.).
Other compounds of Formula I, such as those prepared in accordance with Examples 2-13, can be used as the active compound in the preparation of the suppository formulations of this example.

This example illustrates the preparation of another representative pharmaceutical formulation for oral administration, containing an active compound of Formula I, e.g., 4'-azidothymidine.
Quantity per Inaredients tablet mas.
Active compound 400 cornstarch 50 lactose 145 magnesium stearate 5 The above ingredients are mixed intimately and pressed into single scored tablets.
Other compounds of Formula I, such as those prepared in accordance with Examples 2-13, can be used as the active compound in the preparation of the orally administrable formulations of this example.
07330 (now 1548F) 26530-FF

2C~340~3 Liposome Formulation With 4'-Azidothymidine 5'-mono~hosPhate Sufficient water is added to 100 g of egg-yolk phospholipids to bring the total volume to 1 liter. The mixture is stirred with a homomixer. Then, the mixture is homogenized with an emulsifier under a pressure of 300 kg/cm for 30 minutes, whereby an aqueous phospholipid dispersion is obtained. 4'-Azidothymidine 5~-mono-phosphate (20 g) and sodium chloride (lB g~ are dissolved in enough water to bring the total volume to 1 liter.
The aqueous phospholipid dispersion (850 mL) and the 4'-azidothymidine 5'-monophosphate solution (850 mL) are mixed. The aqueous dispersion thus obtained is filtered through a membrane filter (pore size: 0.45 m in diameter). The filtrate is sterilized at 120C for 20 minutes and then allowed to stand at -20C for 20 hours in a freezer. The frozen dispersion thus obtained is thawed by allowing it to stand at room temperature. An aqueous suspension of 4'-azidothymidine 5'-monophosphate entrapped in phospholipid spherules is thereby obtained.
Other compounds of Formula I, such as those prepared in accordance with Examples 2-13, particularly the phosphate esters, can be used as the active compound in the preparation of formulation according to this example.

Liposome Formulation With 4'-AzidothYmidine 2',3'-diPalmitoate Phosphatidylcholine (30 mM), cholesterol (15 mM) 07330 (now 1548F) 26530-FF

^`` 2cg~40~3 and cholesterol sulfate (5mM) are dissolved in a 2:1 mixture of chloroform:methanol. To this, 4'-azidothymidine 3',5'-dipalmitoate (5mM) is added and the mixture is stirred in a round bottom flask. The solvents are removed by evaporation under reduced pressure to form a film on the inner surface of the flask. The film is dried in vacuo. Saline (2.5 mL3 is added and the solution is shaken under ~2 to swell the film and prepare a lipid suspension. The suspension is sonicated at 10 to 17C for 50 minutes at 20 XHz and 35 W
by a probe-type sonicator under N2. The size of the liposomes obtained range from 22 to 55 mm in diameter.
Other compounds of Formula I, such as the 3',5'-di-adamantoate, preferably the long chain acyl derivatives of Formula I, can be used as the active compound in the preparation of liposomal formulations of this example.

Determination of Activity Utilizing Alex Cells in vitro Assay This procedure is a modification of a procedure initially described by Chen et al., Biochemical PharmacoloaY, 36 (24), 4361-4362 (1987).
A301 (Alex) cells are pre-infected with a constant amount of HIV (LAV Strain) for three hours at 37C. Test compounds and AZT (control) are added to the infected cells in two, three or five-fold dilutions, out four places for screening and eight places for confirmation.
Unless mentioned the highest concentration is 200 mM.
The test plates are incubated in CO2 at 37C for up to twelve days (incubation time depends on virus pool titer) with a partial medium/sample change midpoint through the 07330 (now 1548F) 26530-FF

zca3~0s incubation period. Uninfected A301 cells with sample dilutions, but without virus, are used to evaluate cytotoxicity.
Reverse transcriptase levels are assayed at the end of the incubation period as follows, to determine if antiviral activity is present.
55 mL of medium from the HIV infected Alex cells is added to 10 mL of 3.2% Triton X-100. This solution is incubated for 30 minutes at 37C, then 25 mL of 4X
assay mix is added to it. The 4X assay mix contains t H]TTP, poly(~).oligo(dT), potassium chloride, magnesium chloride, dithiothreitol, and Tris-HCl buffer, pH8Ø After incu~ating this solution for an additional 60 minutes, 20 mL is spotted on Whatman 3 MM paper.
The paper is washed in 5% TCA, 1% sodium pyrophosphate for three times (10 minutes each), followed by one wash (10 minutes) in ethanol. The radioactivity remaining on the 3MM paper is quantitated by scintillation counter, and corresponds to reverse transcriptase level.
The compounds of the present invention show activity when tested by this method, as shown below.

Cell Toxicity Compound EC50 Partial Complete _______________________________________________________ 4'-azidothymidine 0.Ol~M 8rM 200~M
3'-azidothymidine* 0.01~ 825~M 3300 * control 07330 (now 1548F) 26530-FF

2C~3~08 Kinetic Study of Reverse Transcriptase and DNA Polymerase Inhibition This procedure is a modification of procedures initially described by Chen et al., Molecular PharmacoloaY, 25, 441-445 (1984), and as described by Wang et al., BiochemistrY, 21, 1597-1608 (1982).
4'-Azidothymidine 5'-triphosphate was tested as an inhibitor against HIV reverse transcriptase, and against a and o DNA polymerase from human kB cells.
Poly(A)oligo(dT)12_18 was used as a template primer for the HIV reverse transcriptase assay, while activated DNA
was used as a template primer for a and o DNA
polymerases. 4'-Azidothymidine triphosphate was found to be a competitive inhibitor against thymidine triphosphate with a Ki of 0.008 mM, 62.5 mM, and 150 mM for HIV reverse transcriptase, and a and o DNA
polymerases, respectively.

Determination of Toxicity Effects Utilizing Human Hematopoietic Cells in vitro This procedure is a modification of procedures initially described by Diainiak, et al., British Journal of Haematoloav, 69, 229-304 (1988), or as described by Sommadossi, et al., Aqents and Chemo~heraPY, 31 (3), 452-454 (1987).
The effect of 4'-azidothymidine and AZT on the formation of erythroid and GM colonies from precursors in human peripheral blood and bone marrow were compared;
07330 (now 1548F) 26530-FF

~0~3a~08 from which the inhibition of 4'-azidothymidine and AZT
against human hematopoietic cells were compared.

PreParation of Nonadherent Mononuclear Cells In each experiment peripheral blood'or bone marrow from'a single donor was used. Human peripheral blood mononuclear cells (PBL) were separated from heparinized blood of drug-free donors by density-gradient centrifugation in Ficoll (Pharmacia). Whole blood diluted 1:1 with Hanks' balanced salt solution (HBSS, Gibco) was layered on the top of Ficoll and centrifuged at 400 x g for 30 min. Cells were collected from the interphase and after washing, the concentration was adjusted to 2X106 cells/mL in RPMI-1640 medium with 25 mM Hepes buffer (Gibco). Monocytes were removed by adherence to plastic in large Petri dishes (150xl5mm), adding 20 ml of the cell suspension/dish, and incubating for 1 hour at 37C. Nonadherent mononuclear cells (MN) were removed by vigorous washing of the Petri dish with warm (37C) HBSS.

Pre~aration of Bone Marrow Mononuclear Cells (BM) Bone marrow aspirates from healthy donors were collected in heparizined syringes. The whole mononuclear cell fraction was separated by Ficoll-Paque density-gradient centrifugation (400 x g for 30 min).
Cells collected from the interphase were washed 3 times in HBSS and resuspended in culture medium as described below for CFU-GM assay.

CFF-GM AssaY (Mveloid Precursors~
MN (1.5x106 cells/ml) and BM (l.x105 cells/ml) were cultured in complete medium as follows: Dulbecco's Modified Eagle Medium (DMEM, Gibco) supplemented with 20%
heat-inactivated fetal calf serum (FCS, Hyclone), 2 mM
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-2C~3408 L-glutamine, 5 x 10 5 M 2-mercaptoethanol, penicillin (lO0 U/ml), streptomycin (lO0 mg/ml), and 0.3%
bacto-agar (Difco). All supplements were from Gibco, unless otherwise specified. Cells were plated in 35xlO0 mm Lux Petri dishes (suspension dishes) in triplicate, and incubated at 37C with 5% CO2 in air at 100%
humidity. A supernatant from a T-cell line (GCT, Gibco), containing GM-Colony Stimlating Factor (GM-CSF), was used at a final concentration of 10% in complete medium.
Tested compounds were added to the test dishes at concentrations of lO 9 to lO 4 M. Cells were incubated in air ccntaining 5% CO2 (100% humidity) at 37C for 14 days. Colonies were counted under a dissecting microscope. A CFU-GM colony was defined as a cluster of S0 or more cells consisting of granulocytes, monocyte-macrophages, or both.

8FU-E Assay (for earlY ErYthroid Precursors) and CFU-E
AssaY (or Late Er~throid Precursors) MN were cultured at a concentration of 3 x 105 cells/ml. The culture medium consisted of Iscove's Modified Eagle Medium (Gibco) supplemented with 30%
heat-inactiviated FCS (Hyclone), penicillin (lO0 ml), streptomycin (100 mg/ml), 2-mercaptoethanol (1 x lO 4, 1% deionized bovine serum albumin (BSA, Sigma), DEAE-Dextran 40 mg/ml (Pharmacia), 0.8% methylcellulose (4000 Centipose, Fisher), and erythropoietin 2 U/ml (Connaught Laboratories). Cells were plated in 96-well plates (200 ml/well) and incubated at 37C with 5%
C2 in air (100% humidity) for 7-14 days. CFU-E
colonies were counted on Days 7-8. Clusters of 8 or more hemoglobinized cells were considered as a colony. BFU-E
colonies were counted on Day 14. Aggregates of 50 or more cells were counted as a colony. Colonies were confirmed as erythroid by benzidine stainings.
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.'- .
, ~o~o~

Statistical Analvsis All tests were performed in triplicate. The 50%
inhibitor~ concentrations (IC50) for all types of colonies were determined using an RS 1 program on an IBM-PC.
Compounds of the present invention show decreased toxicity when compared with AZT by this method, as shown in the following tables.

Comparative Effects on the Formation of Granulocyte-Macrophage Colonies (CFU-GM) by Human Bone Marrow and Peripheral Blood Mononuclear Cells ___________________________________ ___ _________________ .
Compound IC50 (rM) b PBM BM
Mean ~SEa 1 2 ______________________________________________~____________ 1 20.65 ~1.73 21.09 13.61 2 4.83 ~1.42 2.01 0.90 1 , 4'-azidothymidine 2 , 3'-azidothymidine a , mean ~ SE of five separate experiments are shown b , results trom two separate experiments are presented.

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~ ~ .

:

2C~3408 Comparative Effects on Erythroid Colonies, CFU-E and BFU-E, by Human Bone Marrow and Peripheral Blood Mononuclear Cells _______________________ __________________________________ , C50 t Compound Cells Origin CFU-E BFU-E
Mean Mean ~SD
________________________________________________________ 1 PBM ~3)a ~100 66.57 ~9.23 BM ~1) ~100 NT

2 PBM ~3) ~100 16.02 ~2.98 BM ~1) 26.96 NT

1 z 4'-azidothymidine 2 = 3'=azidothymidine a = the number of experiments performed is indicated in brackets.
NT = not tested.

Determination of Activity Utilizing Friend Leukemia Virus in Mice This procedure is a modification of a procedure initially described by Jones et al., J~urnal of ViroloqY, 62 ~2), 511-518 (1988), or by modifications thereof.
Friend leukemia virus complex (FLVC) was used. This retrovirus complex consists of both helper and defective virus particles.
Six week old female Balb/c mice from Bantin and Kingman, each weighing on the average of 18 gm, received 07330 (now 1548F) 26530-FF

0.2ml of a solution (comprised of virus diluted in P~S
containing 0.002 M EDTA) administered at 20 FFU/mouse, intravenously. Treatments began 4 hours after infection and continued for 9 days. AZT and 4'-azidothymidine were administered at 60 mg/kg/dose twice daily on Days 0, 4, and 5; and on Days 1-3 and 6-8 were administered 3 times daily at 40 mg/kg/dose. 2'3'-Dideosycytidine was administered at 120 mg/kg/dose twice daily on days 0, 4, and 5; and on days 1-3 and 6-8 was administered 3 times daily at 80 mg/kg/dose. All treatments were given intraperitoneally, 0.5ml/dose. On day 9 after infection the spleens were removed, stained and fixed in Bouin's solution for 2 hours and then weighed. Foci were counted on spleens. A standard t-test was run to compare difference in spleen weights and difference in number of foci-forming units (FFU's3 per spleen between groups.
Compounds of the present invention show anti-viral activity when tested by thig method, as shown in the following table.

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2C~)3~08 Efficacy of Compounds Against Friend Leukemia Virus in Mice ___________________._____________________________________ Daily Spleen Inhibition of Dosea Virus Weight SplenomegalyC FFU/
Test Comp'd ~mg/kg) (+/-) (gm)b (%) Spleen __________________________________________________________ 1 1 120 + 0.153d 90 ld 1 120 - 0.119 -- ~ o 2 120 + O.190d 75 ~26 2 120 - 0.104e -- 0 3 0 + 0.435 -- ~29 none none - 0.092 -- 0 2 4 240 + 0.256d 69 ~26 4 240 - 0.138e -- 0 3 0 + 0.473 -- ~ 30 none none - 0.092 -- 0 3 5 240 + 0.211d 68 ~ 16 240 - 0.158e -- 0 1 120 + 0.172d 76 2d 1 120 - 0.132e -- 0 3 0 + 0.265 -- ~ 30 none none - 0.101 00 ~ 30 1 = 3'-azidothymidine 2 = 4'-azidothymidine 3 = saline 4 = 2',3'-dideoxycytidine 5 z 2',3'-dideoxyinosine (ddI) a =i.p. in 2 or 3 equal doses for 9 days starting 4 hr after challenge.
b -weighed on Day 9 after infection.
c =percent inhibition d = _0.05 compared with saline-treated, infected control.
e = ~0.05 compared with untreated, uninfected control.
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40~s While the present invention has been described with reference to the specific embodiments thereof, it should be understood by those skilled in the art that various changes may be made and equivalents may be substituted without departing from the true spirit and scope of the invention. In addition, many modifications may be made to adapt a particular situation, material, composition of matter, process, process step or steps, to the objective, spirit and scope of the present invention. All such modifications are intended to be within the scope of the clairns appended hereto.

07330 (now 1548F) 26530-FF

' :

Claims (22)

1. A compound of the formula wherein:
B is guanine, adenine, thymine, uracil, cytosine, hypoxanthine, xanthine, 5-methylcytosine, 4-ethoxy-5-methyl-2-oxo-pyrimidine, 4-isopropoxy-5-methyl-2-oxo-pyrimidine, or 5-methyl-2-oxo-pyrimidine;
X is H or F;
Y' is H, OH, OCH3 or F; and Z is or where n is zero, one, or three and both R' are hydrogen or lower alkyl; or Y' and Z together form a cyclic phosphate ester;
and the pharmaceutically acceptable esters, ethers and salts thereof.

2. A compound of Claim 1 wherein Y' is OH.

3. A compound of Claim 2 wherein X is H.

07330 (now 1548F) 26530-FF

4. A compound of Claim 3 wherein n is zero, namely 4'-azido-thymidine, 4'-azido-2'-deoxyadenosine, 4'-azido-2'-deoxyguanosine, 4'-azido-2'-deoxyuridine, 4'-azido-2' deoxycytidine, 9-(4-azido-2-deoxy-.beta.-D-erythro-pentofuranosyl)-6-hydroxypurine, 1-(4-azido-2-deoxy-.beta.-D-erythro-pento-furanosyl)-4-amino-5-methyl-2-oxopyrimidine, 1-(4-azido-2-deoxy-.beta.-D-erythro-pento-furanosyl)-4-ethoxy-5-methyl-2-oxopyrimidine, 1-(4-azido-2-deoxy-.beta.-D-erythro-pento-furanosyl)-4-isopropoxy-5-methyl-2-oxopyrimidine, 1-(4-azido-2-deoxy-.beta.-D-erythro-pento-furanosyl)-5-methyl-2-oxopyrimidine, and 9-(4-azido-2-deoxy.beta.-D-erythro-pento-furanosyl)-2,6-dihydroxypurine.

5. A compound of Claim 1 wherein Y' is H.

6. A compound of Claim 5 wherein X is H.

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7. A compound of Claim 6 wherein n is zero, namely 4'-azido-3'-deoxythymidine, 4'-azido-2',3'-dideoxyadenosine, 4'-azido-2',3'-dideoxyuridine, 4'-azido-2',3'-dideoxycytidine, 4'-azido-2',3'-dideoxyguanosine, 9-(4-azido-2,3-dideoxy-.beta.-D-glycero-pentofuranosyl)-6-hydroxypurine, 1-(4-azido-2,3-dideoxy-.beta.-D-glycero-pentofuranosyl)-4-amino-5-methyl-2-oxopyrimidine, 1-(4-azido-2,3-dideoxy-.beta.-D-glycero-pentofuranosyl)-4-ethoxy-5-methyl-2-oxopyrimidine, 1-(4-azido-2,3-dideoxy-.beta.-D-glycero-pentofuranosyl)-4-isopropoxy-5-methyl-2-oxopyrimidine, 1-(4-azido-2,3-dideoxy-.beta.-D-glycero-pentofuranosyl)-5-methyl-2-oxopyrimidine, and 9-(4-azido-2,3-dideoxy-.beta.-D-glycero-pentofuranosyl)-2,6-dihydroxypurine.

8. A compound of Claim 1 wherein Y' is F.

9. A compound of Claim 8 wherein X is H.

10. A compound of Claim 9 wherein n is zero.

11. A compound of Claim 4 wherein n is one or three.

12. A compound of Claim 6 wherein n is one or three.

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13. A compound of Claim 9 wherein n is one or three.

14. A pharmaceutical composition comprising a pharmaceutically acceptable non-toxic excipient and therapeutically effective amount of a compound of Formula 1 wherein:
B is guanine, adenine, thymine, uracil, cytosine, hypoxanthine, xanthine, 5-methylcytosine, 4-ethoxy-5-methyl-2-oxo-pyrimidine, 4-isopropoxy-5-methyl-2-oxo-pyrimidine, or 5-methyl-2-oxo-pyrimidine;
X is H or F;
Y' is H, OH, OCH3 or F; and Z is or where n is zero, one, or three and both R' are hydrogen or lower alkyl; or Y' and Z together form a cyclic phosphate ester;
or a pharmaceutically acceptable ester, ether or salt thereof.

15. The pharmaceutical composition of Claim 14 comprising a therapeutically effective amount of a second antiviral agent.

07330 (now 1548F) 26530-FF

16. The pharmaceutical composition of Claim 15 where said second antiviral agent is acyclovir, ganciclovir or foscarnet.

17. The pharmaceutical composition of Claim 14, wherein the compound is 4'-azidothymidine.

18. A compound of the formula wherein:
B' is guanine, adenine, thymine, uracil, cytosine, hypoxanthine, xanthine, 5-methylcytosine, 4-ethoxy-5-methyl-2-oxo-pyrimidine, 4-isopropoxy-5-methyl-2-oxo-pyrimidine, or 5-methyl-2-oxo-pyrimidine or an acylated equivalent thereof;
R is methyl or an acyl group;
X is H or F; and Z is iodomethyl or bromomethyl.

19. A compound of Claim 18 wherein Z is iodomethyl.

20. A compound of Claim 19 wherein R is anisoyl, benzoyl, acetyl or furan-2-carbonyl.

07330 (now 1548F) 26530-FF

21. A process for preparing a compound of formula I

.
wherein:
B is guanine, adenine, thymine, uracil, cytosine, hypoxanthine, xanthine, 5-methylcytosine, 4-ethoxy-5-methyl-2-oxo-pyrimidine, 4-isopropoxy-5-methyl-2-oxo-pyrimidine, or 5-methyl-2-oxo-pyrimidine;
X is H or F;
Y' is H, OH, OCH3 or F; and Z is or where n is zero, one, or three and both R' are hydrogen or lower alkyl; or Y' and Z together form a cyclic phosphate ester;
and the pharmaceutically acceptable esters, ethers and salts thereof, which comprises a) reacting a compound of formula II

Formula II
wherein:
B' is the same as B in Formula I, or an acylated 07330 (now 1548F) 26530-FF

equivalent thereof;
R is methyl or an acyl group such as anisoyl, benzoyl, acetyl or furan-2-carbonyl;
X is H or F; and Z is methylene with a leaving group such as iodo or bromo;
with an oxidizing agent, followed by the addition of a base; or b) reacting a compound of the formula wherein:
B is guanine, adenine, thymine, uracil, cytosine, hypoxanthine, xanthine, 5-methylcytosine, 4-ethoxy-5-methyl-2-oxo-pyrimidine, 4-isopropoxy-S-methyl-2-oxo-pyrimidine, or 5-methyl-2-oxo-pyrimidine;
X is H or F;
Y' is H, OH, OCH3 or F;
R3 is lower alkyl; and R4 is -OH or tri-substituted silyloxy;

with (1) if R4 is OH, a silylating agent and a base followed by an azide in the presence of a Lewis acid catalyst, or (2) if R4 is tri-substituted silyloxy, reacting with an azide in the presence of a Lewis acid catalyst; or c) reacting a compound of formula I wherein n is zero, with a phosphorylating agent to give a compound of 07330 (now 1548F) 26530-FF

formula I wherein n is one or three; or d) reacting a compound of formula I wherein n is one with a cyclizing agent to give a compound of formula I wherein Y' and Z together form a cyclic phosphate; or e) reacting a compound of formula II with phosphonous acid or a salt of a di-lower alkyl phosphonate to form a compound of formula I wherein z is (R'O)2P(O)CH2-; or f) reacting a compound of formula I with a protecting agent, followed by reaction with a strong base followed by reaction with an alkyl halide, followed by deprotection, to give a compound of formula I wherein Y' is an ether; or g) reacting a compound of formula I with a pyridine catalyst and an acid chloride to give the corresponding ester of a compound of formula I; or h) reacting a compound of formula I with a pharmaceutically acceptable base to form the corresponding base addition salt of formula I; or i) reacting a base addition salt of a compound of formula I with an acid to form the free compound of formula I; or j) converting a pharmaceutically acceptable base addition salt of a compound of formula I to another pharmaceutically acceptable base addition salt of a compound of formula I; or k) converting an ester or ether of a compound of formula I to the corresponding free compound of formula I.

22. A method of treating a mammal for any infection of the group consisting of viral, bacterial or fungal infections, said method comprising administering to a mammal in need thereof a therapeutically effective amount of a 07330 (now 1548F) 26530-FF

compound represented by the formula wherein:
B is guanine, adenine, thymine, uracil, cytosine, hypoxanthine, xanthine, 5-methylcytosine, 4-ethoxy-5-methyl-2-oxo-pyrimidine, 4-isopropoxy-5-methyl-2-oxo-pyrimidine, or 5-methyl-2-oso-pyrimidine;
X is H or F;
Y' is H, OH, OCH3 or F; and Z is or where n is zero, one, or three and both R' are hydrogen or lower alkyl; or Y' and Z together form a cyclic phosphate ester;
or a pharmaceutically acceptable ester, ether or salt thereof.

07330 (now 1548F) 26530-FF